**2. Mechanisms of action of antimicrobial agents**

There are six basic mechanisms of antimicrobial agents presented below:

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important barrier.

*Chemotherapy and Mechanisms of Action of Antimicrobial Agent*

*DOI: http://dx.doi.org/10.5772/intechopen.95476*

3.Inhibition of microbial protein synthesis.

4.Inhibition of microbial DNA synthesis.

5.Inhibition of microbial RNA synthesis.

**2.1 Inhibition of microbial cell wall synthesis**

6.Inhibition of microbial metabolic pathways.

An integral microbial structure responsible for the shape of the cell is the cell wall. In addition, because of the high cytoplasmic osmotic pressure, the cell wall prevents cell lysis and facilitates the anchoring of membrane components and extracellular proteins, such as adhesins [25]. Bacterial cell wall synthesis has perhaps become the target field most commonly exploited for antimicrobial production on the basis of the number of antimicrobial drugs in clinical usage. Due to the absence of equivalents in human biology, the components of the cell wall synthesis machinery are attractive antimicrobial targets, thus providing intrinsic objective selectivity. The cytoplasmic synthesis of building blocks composed of N-acetyl muramic acid (M) linked to N-acetyl glucosamine (G) with an attached pentapeptide (P) side chain (referred to as MGP subunits) comprises the sequential late steps in cell wall synthesis. The linkage of the MGP subunit to the lipid II molecule enables subsequent translocation to the outside or periplasmic space of the cell through the cytoplasmic membrane. By catalyzing glycosidic linkages between the M and G components of the MGP subunits, transglycosylase enzymes then assemble the MGP subunits into a linear backbone. An immature peptidoglycan structure is constituted by linearly connected MGP subunits. Transpeptidase enzymes then work to cross-link pentaglycine bridges to the peptide side chains, in the process, the terminal 2 D-alanines of the peptide side chain are cleaved, creating the mature, lattice-like peptidoglycan that provides the form and osmotic stability of the bacterium [26]. β-lactam antibiotics, such as penicillins and cephalosporins, are the most widely used antimicrobials that prevent cell wall biosynthesis [27]. These β-lactam antibiotics interact directly with bacterial transpeptidases and inhibit them effectively. As transpeptidase inhibitors, β-lactams thus obstruct the transition from immature to mature peptidoglycan, so these enzymes are also referred to as penicillin-binding proteins (PBPs). Due to the stereochemical similarity of the β-lactam moiety with the D-alanine-D-alanine substrate, they are capable of doing this. Transpeptidases form a lethal covalent penicilloyl enzyme complex in the presence of the drug, which helps to inhibit the usual transpeptidation reaction. This results in peptidoglycan that is weakly cross-linked, which makes the

developing bacteria extremely susceptible to cell lysis and death [28].

Lipids, proteins and lipoproteins are essentially made of biological membranes. The cytoplasmic membrane for water, ions, nutrients and transport systems serves as a diffusion barrier. Most health workers now assume that membranes are a lipid matrix with uniformly distributed globular proteins to penetrate through the bilayer of the lipid. A number of antimicrobial agents may cause disorganization of the membrane. These agents can be categorized into cationic, anionic, and neutral agents. Polymyxin B and colistemethate (polymyxin E) are the best-known compounds [29]. For several antimicrobial agents, the cytoplasmic membrane forms an

**2.2 Inhibition of microbial cell membrane function**


*Chemotherapy and Mechanisms of Action of Antimicrobial Agent DOI: http://dx.doi.org/10.5772/intechopen.95476*


*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

of empirical antibiotic therapy against this microorganism [8]. Resistance to a range of antibiotics, including aminoglycosides, quinolones and β-lactams, is demonstrated by *P. aeruginosa* [9]. Generally, the main mechanisms of *P. aeruginosa* used to fight antibiotic attack can be divided into intrinsic, acquired and adaptive resistance. Low external membrane permeability, the expression of efflux pumps that remove antibiotics from the cell, and the development of antibiotic inactivating enzymes are part of the intrinsic resistance of *P. aeruginosa*. Either horizontal transfer of resistance genes or mutational modifications will achieve the acquired resistance of *P. aeruginosa* [10]. *P. aeruginosa 's* adaptive resistance requires the development of biofilm in the lungs of infected patients, where the biofilm functions as a diffusion barrier to inhibit the access of antibiotics to bacterial cells [11]. The effectiveness and safety of murepavadin in the treatment of infections of the lower respiratory tract caused by *P. aeruginosa* (suspected or confirmed) in patients with ventilation-associated pneumonia or CF-unrelated bronchiectasis (Clinical Trials. gov identifiers NCT02096315 and NCT02096328) have been tested in two clinical trials. However, by July 17, 2019, the trials were stopped because in research participants who had obtained murepavadin, an unusually high level of renal failure had been found. This decision would not impact the production of an aerosolized formulation of murepavadin for topical use [12]. Murepavadin is a particular weapon against *P. aeruginosa*, which separates it from the broad pipeline of antimicrobial natural and synthetic peptides acting against multiple taxa, *P. aeruginosa* included. Recently, several novel peptides with broad antimicrobial activity have been identified, such as antimicrobial peptide DGL13K, Mel4 and melamine (Melimine and Mel4 are chimeric cationic peptides with broad-spectrum antimicrobial activity), Cecropin B, Lysine-based peptidomimetics (LBP-2), Truncated pseudin-2 analogs (Pse-T2), antimicrobial peptide, termed 6 K-F17 (sequence: KKKKKK-AAFAAWAAFAA-NH2), Melittin-derived peptides (MDP1, MDP2) [13–20]. In addition, multidrug-tolerant persistent cells can form in the biofilm that are capable of surviving antibiotic attack; in cystic fibrosis (CF) patients, these cells are responsible for prolonged and recurrent infections [21]. For patients whose infections are resistant to traditional antibiotics, the development of new antibiotics or alternative therapeutic methods for treating *P. aeruginosa* infections is urgently needed. In recent years, new antibiotics with novel modes of action have been investigated, as have new routes of administration and resistance to bacterial enzyme alteration. Compared to traditional antibiotics, some of these newer antibiotics demonstrate excellent in vitro antibacterial activity against *P. aeruginosa* as well as lower minimum inhibitory concentration (MIC) [22, 23]. Moreover, several novel non-antibiotic therapeutic approaches that are highly successful in destroying antibiotic resistant *P. aeruginosa* strains have been documented in recent studies [24]. These approaches include: antimicrobial peptides, phage therapy, inhibition of quorum sensing, iron chelation, the use of nanoparticles, probiotic and vaccine strategy. In order to combat *P. aeruginosa* infections, these therapeutic approaches may be used either as an alternative to or in conjunction with traditional antibiotic

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therapies.

**2. Mechanisms of action of antimicrobial agents**

2.Inhibition of microbial cell membrane function.

1.Inhibition of microbial cell wall synthesis.

There are six basic mechanisms of antimicrobial agents presented below:


## **2.1 Inhibition of microbial cell wall synthesis**

An integral microbial structure responsible for the shape of the cell is the cell wall. In addition, because of the high cytoplasmic osmotic pressure, the cell wall prevents cell lysis and facilitates the anchoring of membrane components and extracellular proteins, such as adhesins [25]. Bacterial cell wall synthesis has perhaps become the target field most commonly exploited for antimicrobial production on the basis of the number of antimicrobial drugs in clinical usage. Due to the absence of equivalents in human biology, the components of the cell wall synthesis machinery are attractive antimicrobial targets, thus providing intrinsic objective selectivity. The cytoplasmic synthesis of building blocks composed of N-acetyl muramic acid (M) linked to N-acetyl glucosamine (G) with an attached pentapeptide (P) side chain (referred to as MGP subunits) comprises the sequential late steps in cell wall synthesis. The linkage of the MGP subunit to the lipid II molecule enables subsequent translocation to the outside or periplasmic space of the cell through the cytoplasmic membrane. By catalyzing glycosidic linkages between the M and G components of the MGP subunits, transglycosylase enzymes then assemble the MGP subunits into a linear backbone. An immature peptidoglycan structure is constituted by linearly connected MGP subunits. Transpeptidase enzymes then work to cross-link pentaglycine bridges to the peptide side chains, in the process, the terminal 2 D-alanines of the peptide side chain are cleaved, creating the mature, lattice-like peptidoglycan that provides the form and osmotic stability of the bacterium [26]. β-lactam antibiotics, such as penicillins and cephalosporins, are the most widely used antimicrobials that prevent cell wall biosynthesis [27]. These β-lactam antibiotics interact directly with bacterial transpeptidases and inhibit them effectively. As transpeptidase inhibitors, β-lactams thus obstruct the transition from immature to mature peptidoglycan, so these enzymes are also referred to as penicillin-binding proteins (PBPs). Due to the stereochemical similarity of the β-lactam moiety with the D-alanine-D-alanine substrate, they are capable of doing this. Transpeptidases form a lethal covalent penicilloyl enzyme complex in the presence of the drug, which helps to inhibit the usual transpeptidation reaction. This results in peptidoglycan that is weakly cross-linked, which makes the developing bacteria extremely susceptible to cell lysis and death [28].

#### **2.2 Inhibition of microbial cell membrane function**

Lipids, proteins and lipoproteins are essentially made of biological membranes. The cytoplasmic membrane for water, ions, nutrients and transport systems serves as a diffusion barrier. Most health workers now assume that membranes are a lipid matrix with uniformly distributed globular proteins to penetrate through the bilayer of the lipid. A number of antimicrobial agents may cause disorganization of the membrane. These agents can be categorized into cationic, anionic, and neutral agents. Polymyxin B and colistemethate (polymyxin E) are the best-known compounds [29]. For several antimicrobial agents, the cytoplasmic membrane forms an important barrier.

The mode of action of certain antimicrobial agents may be due to the ability of such medicines to increase membrane permeability, making it easier for them and other compounds to penetrate. Antibacterial cationic agents, increased permeability of the outer membrane to the lysozyme and hydrophobic compounds has been identified, such as polymyxin B. The initial function of these antimicrobial agents is to interrupt the structure of the outer membrane, allowing the cell to join itself and other compounds and inhibit unique metabolic processes [30]. There are several cell-damaging properties of Polymixin B: (i) the surface charge, lipid composition and membrane structure are disturbed; (ii) the K+ gradient on the cytoplasmic membrane is dissipated; and (iii) the cytoplasmic membrane is depolarized. One of the key factors regulating bacterial exposure to polymixin B is the permeability of the external membrane to lipophilic compounds. Since polymixin B is bulkier than its displacement of inorganic divalent cations, in the presence of polymixin B, the packing order of lipopolysaccharides (LPS) is changed. This results in increased permeability of a variety of molecules to the outer membrane and also promotes polymixin B uptake ("self-promoted" uptake) [31].

#### **2.3 Inhibition of microbial protein synthesis**

Microbial protein synthesis inhibition a range of groups of antimicrobial agents work by inhibiting the synthesis of bacterial proteins (ribosome function). That include aminoglycosides, macrolides, tetracyclines, ketolides, lincosamides, streptogramins, chloramphenicol and oxazolidinones [26, 32]. The synthesis of microbial proteins is led by ribosomes in conjunction with cytoplasmic factors which, during the initiation phase, elongation phase and termination phases, bind transiently to particles. Microbial ribosomes contain 70S particles consisting of two 50S and 30S subunits, which join at the initiation stage of the synthesis of proteins and split at the termination stage. In bacterial protein synthesis, antimicrobial agents block various steps by interfering with the work of either the cytoplasmic factors or the ribosomes. Inhibitors which bind to the ribosomal subunit of 30S primarily interfere with initiation, although some interfere with the pairing of the AA- tRNA anticodon with the mRNA codon, elongation is thus impaired. The steps involved in the elongation process interact with inhibitors that bind to the 50S ribosomal subunit or to elongation factors that are transiently connected to ribosomes at certain stages of the cycle.

Through binding to particular ribosomal subunits [33], aminoglycosides function. By inducing the development of aberrant, non-functional complexes as well as causing misreading, aminoglycoside-type drugs may combine with other binding sites on 30S ribosomes and destroy bacteria. Spectinomycin is an antimicrobial agent that is closely linked to the aminoglycosides of aminocylitol. It binds and is bacteriostatic but not bactericidal to a particular protein in the ribosome. Tetracyclines are other agents which bind to 30S ribosomes. These agents tend to inhibit aminoacyl tRNA binding to the A site of the bacterial ribosome. Tetracycline binding is temporary, so it's bacteriostatic for these agents. Nevertheless, a wide range of bacteria, *chlamydias* and *mycoplasmas* are inhibited and highly helpful agents [29]. There are three major groups of medicines that inhibit the ribosomal subunit of 50S. A bacteriostatic agent that inhibits both gram-positive and gram-negative bacteria is chloramphenicol. By binding to a peptidyltransferase enzyme on the 50S ribosome, it prevents peptide bond formation. Macrolides are large compounds of the lactone ring that bind to 50S ribosomes and tend to impair the reaction or translocation of peptidyltransferase, or both. Erythromycin, which inhibits gram-positive species and a few gram-negative species, such as *haemophilus, mycoplasma, chlamydia* and

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compounds [37].

to be discovered as such [36].

**2.5 Inhibition of microbial RNA synthesis**

*Chemotherapy and Mechanisms of Action of Antimicrobial Agent*

only inhibit the development of new peptide chains [29].

*legionella*, is the most significant macrolide. Against many of these pathogens, new molecules including azithromycin and clarithromycin have greater activity than erythromycin. There is a similar activity site for lincinoids, the most important of which is clindamycin. Generally, macrolides and lincinoids are bacteriostatic and

The modulation of chromosomal supercoiling by topoisomerase-catalyzed strand breakage and rejoining reactions is needed for DNA synthesis, mRNA transcription and cell division [34]. Depending on whether they catalyze reactions involving transient breakage of one (type I) or both (type II) strands of DNA, DNA topoisomerase enzymes are classified into two groups, I and II [35]. The topological state of DNA inside cells is regulated by topoisomerases and is important for the vital processes of protein translation and cell replication. The enzyme that negatively super-coils DNA in the presence of ATP is DNA gyrase, a type II DNA topoisomerase [36]. Moreover, in the absence of ATP, this enzyme plays a role in the catenation and decatenation reaction of a double-stranded DNA circle, resolves knots in DNA, and also relaxes supercoiled DNA negatively. As a result, for almost all cellular procedures involving duplex DNA, including replication, recombination and transcription, the enzyme is vital. It is unique to the prokaryotic kingdom and is essential to the organism's survival. Thus, for antibacterial drugs, DNA gyrase remains an ideal and attractive target. The most effective DNA gyrase-targeted antimicrobial agents are quinolones. Nalidixic acid, a naphthyridone inadvertently discovered as a by-product during chloroquine synthesis, was the source of the

Quinolones are unique DNA-gyrase inhibitors. DNA gyrase reactions such as supercoiling and relaxation involving DNA breakage and reunion are inhibited by quinolones, specifically interfering with the DNA gyrase breakage-reunion reaction by interacting with subunit A (GyrA) [38]. Relatively poor antimicrobial activity is found in first-generation quinolones, nalidixic acid and oxolinic acid. However, the synthesis and improvement over many generations of fluoroquinolones, such as norfloxacin and ciprofloxacin (second generation), levofloxacin (third generation), and moxifloxacin and gemifloxacin (fourth generation), has resulted in a variety of potent antimicrobial agents [38]. Most bacterial pathogens possess an additional essential topoisomerase, topoisomerase I (Topo I), in addition to the type II topoisomerases. Topo I is architecturally and mechanistically distinct from gyrase and topoisomerase IV, and is an attractive candidate for new antibacterial chemotypes

Rifamycins inhibit DNA-dependent transcription by binding the DNA-bound and effectively transcribing RNA polymerase with a high affinity to the β-subunit (coded by rpoB). In the channel formed by the RNA polymerase-DNA complex, from which the newly synthesized RNA strand emerges, the β- subunit is located. Rifamycins clearly require that RNA synthesis has not progressed beyond two ribonucleotides being added; This is due to the drug molecule 's capacity to sterically inhibit the initialization of nascent RNA strands. It should be noted that rifamycins are not believed to work by blocking the RNA synthesis elongation stage, although a recently discovered class of RNA polymerase inhibitors (based on the CBR703 compound) could inhibit elongation by modifying the enzyme allosterically [34].

*DOI: http://dx.doi.org/10.5772/intechopen.95476*

**2.4 Inhibition of microbial DNA synthesis**

*legionella*, is the most significant macrolide. Against many of these pathogens, new molecules including azithromycin and clarithromycin have greater activity than erythromycin. There is a similar activity site for lincinoids, the most important of which is clindamycin. Generally, macrolides and lincinoids are bacteriostatic and only inhibit the development of new peptide chains [29].
