**3.2 AmpC β-lactamase (Cephelosporinase)**

In particular, the development of endogenous β-lactamase, such as chromosomal cephalosporinase (AmpC β-lactamase). A variety of β-lactams, such as benzyl penicillin, narrow spectrum cephalosporin and imipenem, can be induced in *P. aeruginosa*. Naturally, *P. aeruginosa* is susceptible to carboxypenicillins, ceftazidime and aztreonam, but it can develop resistance through a mutation in the gene that contributes to AmpC β-lactamase hyper-production [52, 53]. The enzyme is usually produced in small amounts ('low-level' expression), resistance to aminopenicillins and to most early cephalosporins is determined. *P. aeruginosa* produces an inducible chromosomecoded AmpC β-lactamase (cephalosporinase) belonging to the Ambler-based molecular class C and the first functional group according to Bush et al. [54, 55].

However, production of chromosomal cephalosporin in *P. aeruginosa*, in the presence of inducing β-lactams (especially imipenem), can increase from 100 to 1000 times [56]. β-lactamase inhibitors used in clinical practice, such as clavulanic acid, sulbactam and tazobactam, do not inhibit AmpC cephalosporinase function. β-lactamase of AmpC is encoded by the gene ampC [57, 58]. Several genes, including ampR, ampG, and ampD, are involved in ampC gene induction. AmpR encodes a positive transcriptional regulator and this regulator is required for the induction of β-lactamase. AmpG, a transmembrane protein that functions as a permease for 1,6-anhydromurapeptides, which are known to be the signal molecules involved in the induction of ampC, is the second gene involved. The third gene, ampD, encodes a cytosolic amidase of N-acetyl-anhydromuramyl-L-alanine that hydrolyses 1,6-anhydromurapeptides, which functions as an ampC expression repressor. The 4th chromosome, ampE, encodes the protein of the cytoplasmic membrane that serves as the molecule of the sensory transducer necessary for induction. Except for avibactam, the activity of this AmpC β-lactamase is not inhibited by commercially available β-lactam.

#### **3.3 Class A carbenicillin hydrolysing β-lactamases**

Four β-lactamases (PSE- of *Pseudomonas* specific enzyme) carbenicillin hydrolyzing enzymes were identified in *P. aeruginosa*: PSE-1 (CARB-2), PSE-4 (CARB-1), CARB-3 and CARB-4 [59]. These enzymes belong to the group of β-lactamases of molecular class A and include carboxypenicillins, ureidopenicillins and cefsulodine in their substrate profile. These enzymes belong to functional group 2c and molecular class A [60]. Commercially available β-lactam inhibitors, such as clavulanic acid, tazobactam, and sulbactam, can inhibit the activity of this β-lactamase [61].

#### **3.4 Resistance to aminoglycoside**

Aminoglycosides are a microbial protein synthesis inhibitor which act by binding to the ribosomal subunit of the bacterial 30S and interfering with the initiation of protein synthesis. Resistance to aminoglycosides in *Pseudomonas* is mediated by

transferable aminoglycoside modifying enzymes (AMEs), low permeability of the outer membrane, active efflux and, in rare cases, target modification [62–64].

#### **3.5 Aminoglycoside-modifying enzymes**

AMEs inactivate the aminoglycoside by adding the antibiotic molecule to a phosphate, adenyl or acetyl radical, and thus modified antibiotics minimize the binding affinity of the bacterial cell (30S ribosomal subunit) to its target [65, 66]. Aminoglycoside phosphoryl transferases (APHs), aminoglycoside adenylyl transferases (also known as nucleotidyltransferases) (AADs or ANTs) and aminoglycoside acetyltransferases (AACs) are three types of AMEs involved in aminoglycoside alteration. The following AMEs are most commonly expressed by *P. aeruginosa:* AAC(69)-II (resistant to gentamicin, tobramycin and netilmicin), AAC(3)-I (resistant to gentamicin), AAC(3)-II (resistant to gentamicin, tobramycin and netilmicin), (69)-I (resistant to tobramycin, netilmicin and amicacin) and ANT(29)-I (resistant to tobramicin and gentamicin) [67].

#### **3.6 Low outer membrane permeability**

Membrane impermeability or reduced permeability is a mechanism known to provide resistance to many antibiotic forms, including aminoglycosides, β-lactams and quinolones [68]. For instance, this resistance mechanism is often encountered in cystic fibrosis isolates that are continually under antibiotic attack. Several mechanisms, such as lipopolysaccharide (LPS) modifications, alteration of membranous proteins involved in substratum absorption, and inactivation of enzymatic complexes involved in the energetic membrane necessary for transport system activity, may cause membrane impermeability [69].

#### **3.7 Active efflux pumps**

The combination of low membrane permeability and active efflux pumps is partially due to the natural resistance of *P. aeruginosa* to many groups of antibiotics. *P. aeruginosa's* efflux systems involved in antibiotic resistance belong to the family of resistance-nodulation-division (RND) [70]. In order to confer resistance to several antibiotics, four major efflux systems have been described: MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM. These systems consist of three proteins: (1) the efflux pump protein found in the cytoplasmic membrane (MexB, MexD, MexF and MexY), (2) the pore-acting outer membrane protein (OprM, OprJ and OprN) and (3) A protein in the periplasmic space that bridges the cytoplasmic and outer membrane proteins (MexA, MexC, MexE and MexX). In both natural and acquired resistance, MexAB-OprM and MexXY-OprM are active, whereas only the other two mechanisms are observed in cumulative resistance.

Acquired resistance is observed following mutations in the regulatory systems that can be caused by antibiotic pressure and that can confer resistance to all groups of antibiotics upon over-expression of these efflux systems. Polymyxins, except [69]. Resistance to multiple groups of antibiotics that are substrates of these efflux systems can be caused by exposure to a single antibiotic. Quinolones are substrates of all efflux systems and are an important trigger factor that can generate cross-resistance to efflux systems of several major classes of antibiotics, including β-lactams and aminoglycosides, for pseudomonal therapy [71]. It is understood that efflux systems confer a moderate degree of resistance, but they typically act simultaneously with other mechanisms of resistance, thus taking part in the high-level resistance that can be observed in *P. aeruginosa.*

**83**

*Chemotherapy and Mechanisms of Action of Antimicrobial Agent*

Due to the low affinity of the drug to the bacterial ribosome, bacteria may be resistant to aminoglycosides. This can be achieved by 16S rRNA methylation by target modification. Various 16S rRNA methylases have been identified for *P. aeruginosa*: RmtA, first reported in clinical isolates of *P. aeruginosa* resistant to aminoglycosides and conferred resistance to all parenterally administered aminoglycosides, including amicacin, tobramycin, isepamicin, kanamycin, arbecacin and gentamicin, secondary 16S rRNA methylases including RmtB, ArmA and RmtD [72].

Resistance to fluoroquinolones arises by mutation in the DNA gyrase or topoisomerase 1 V coding bacterial chromosome gene or by successful drug transport out of the cell [73]. Topoisomerase 1 V mutations can occur in gyrA / gyrB genes within the motif of the quinolone-resistant determinative region (QRDR), which is considered to be the active site of the enzyme. This contributes to the altered amino acid sequences of the subunits A and B, and hence to the altered topoisomerase II with a low affinity for quinolone molecules. As a result of point mutations in parC and parE genes encoding the ParC and ParE enzyme subunits, modifications of a secondary target (topoisomerase IV) occur. The over-expression of efflux includes other types of fluoroquinolone tolerance in *Pseudomonas*. Mutations in the nalB, nfxB and nfxC genes, resulting in overexpression of MexA-MexB-OprM,

A biofilm is an aggregate of microorganisms that bind to each other on a living or non-living surface and are embedded in an extracellular polymeric (EPS) matrix of self-produced substances, including exopolysaccharides, proteins, metabolites, and eDNA [75, 76]. The microbial cells grown in biofilms are less sensitive than the cells grown in free aqueous suspension to the antimicrobial agents and the host immune response [77]. Even bacteria that are deficient or lack protective mutations in their intrinsic resistance, when they grow in a biofilm, they can become less susceptible to antibiotics [78]. The general mechanisms of biofilm-mediated resistance that protect bacteria from antibiotic attack include antibiotic penetration prevention, altered microenvironment that induces slow biofilm cell growth, adaptive stress response induction, and differentiation of persistent cells [78–80]. *P. aeruginosa* causes chronic lung infections in CF patients and, through the production of DNA, proteins and exopolysaccharides, forms a biofilm on lung epithelial cell surfaces. The regulation of the formation of *P. aeruginosa* biofilm is multifactorial and mainly depends on quorum sensing systems, GacS / GacA and RetS / LadS two-component regulatory systems, exopolysaccharides and cdi- GMP [81]. Quorum sensing is a form of communication between bacterial cells and cells that regulates gene expression in response to changes in cell population density. *P. aeruginosa* has three major systems of quorum sensing, LasILasR, RhlI-RhlR, and PQS-MvfR, all of which contribute to mature and differentiated biofilm formation. During biofilm formation, *P. aeruginosa* undergoes numerous physiological and phenotypic changes [82]. For example, *P. aeruginosa* strains convert to a mucoid phenotype in CF chronic infection that displays upregulated production of alginate driven by the CF microenvironment, enabling the formation of colonies of biofilms. Due to its ability to show swarming and twitching motility, *P. aeruginosa* flagellum is important for the initiation of biofilm formation. However, *P. aeruginosa* significantly

MexC-MexD- OprJ and MexE- MexF- OprN fallowing efflux [74].

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

**3.9 Resistance to fluoroquinolones**

**3.10 Biofilm-mediated resistance**

**3.8 Target modification**
