*4.1.2.2. Target modification*

genera, in particular between gram-positive and gram-negative bacteria [35]. Plasmid-en‐ coded antibiotic resistance encompasses most classes of antibiotics in practice, such as ami‐ noglycosides, cephalosporins and fluoroquinolones [36]. Transposons spread quicker than genes in chromosomes and are transferred by conjugation, transformation, or transduction [23; 24]. Integrons acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism. They can integrate stably into other DNAs where they deliver multiple antibacterial resistant genes in a single exchange. Resistance gene cas‐ settes encoding the metallo-β- lactamases IMP and VIM confer resistance to the potent car‐

As so many scientists have been struggling to study the biochemical mechanisms of antibi‐ otic resistance, nowadays there is a large pool of related valuable information left. Biochemi‐ cal mechanisms may be varied among different bacterial species, but can be mainly classified into four categories (Fig. 2). In fact, each of these four categories also contains an amazing diversity of resistance mechanisms. Sometimes a single bacterial strain may pos‐ sess several types of resistance mechanisms. Each of the four main categories will be dis‐

Biochemical strategies include enzymatic modification and redox mechanisms (which is less important and will not be elaborated in this paper). Enzymes can be divided into two gener‐ al classes: those such as β-lactamases that degrade antibiotics and others that perform chem‐ ical transformations. The antibiotic β-lactam has a four-atom ring known as a beta-lactamin. The β-lactamase enzyme breaks that ring open, destroying the antibacterial properties of the drugs. β-lactamase consists of enzymes with a serine residue at the active site, and metal‐ loenzymes with zinc ion as a cofactor and with a separate heritage [37]. β-lactamase en‐ zymes are the most common and important weapons for Gram-negative bacteria to resist the antibiotics β-lactam [38]. The group transfer approaches are the most diverse and in‐ clude the modification by acyltransfer, phosphorylation, glycosylation, nucleotidylation, ri‐ bosylation, and thiol transfer. They can inactivate antibiotics (aminoglycosides, chloramphenicol, streptogramin, macrolides or rifampicin) by chemical substitution. These modifications reduce the affinity of antibiotics to a target [85]. For example, enzymatic mod‐ ification is the most prevalent mechanism to destroy aminoglycosides in clinic. Aminoglyco‐ side modifying enzymes can be divided into three classes: acetyltransferases, nucleotidyltranferases, and phosphotransferases; they mainly catalyze the modification at – OH or –NH2 groups of the 2-deoxystreptamine nucleus or the sugar moieties [39]. There are a large number of genes in the chromosomes and other mobile genetic elements coding for these enzymes which let the bacteria resist to more new antibiotics as well as horizontally spread their resistance among bacteria more easily. As a consequence, almost all pathogens

are resistant to aminoglycosides through modifying enzymes [39].

bapenem β-lactams imipenem and meropenem [36].

*4.1.2. Biochemistry of antibiotic resistance*

cussed respectively below.

294 Drug Discovery

*4.1.2.1. Antibiotic inactivation*

Another important resistance mechanism is the modification of antibiotic targets which makes the antibiotic unable to bind the targets properly. β-lactams target the bacterial en‐ zymes of cell wall biosynthesis (the so-called penicillin-binding proteins, PBPs). Alterations in PBPs can reduce affinity for β-lactams, possibly causing β-lactam resistance in many bac‐ teria strains, such as H. influenzae, N. gonorrhoeae, N. meningitidis, anaerobes, S. dysenter‐ iae [40]. For instance, the mecA resistance gene which encodes PBP2a, a new penicillin binding protein with decreased affinity for oxacillin and most other β-lactam drugs, induces resistance to methicillin and oxacillin in S. aureus [41]. The resistance to antibiotics that in‐ terfere with protein synthesis or transcription is achieved by modification of the specific tar‐ get. rRNAmethylases encoded by a number of genes modificate the 16S rRNA molecule at specific positions critical for aminoglycosides binding [42]. Modification in the 23S rRNA component of the 50S ribosomal subunit also leads to resistance to the macrolide, lincosa‐ mide and streptogramin B group of antibiotics in many pathogen strains [43; 44]. Mutations of topoisomerase IV and gyrase genes can sufficiently alter affinity of fluoroquinolones to these enzymes [45].

#### *4.1.2.3. Efflux pumps and outer membrane (OM) permeability*

*Efflux pumps* Membrane proteins that export antibiotics from the cell and maintain their low intracellular concentrations are called efflux pumps. Drug efflux pumps play a key role in drug resistance not just because they can produce multidrug resistance but also because they can elevate level of other resistance mechanisms [46; 47]. Bacterial drug efflux trans‐ porters are currently classified into five families: the ATP-binding cassette (ABC) superfami‐ ly, the major facilitator superfamily (MFS), the multidrug and toxic compound extrusion (MATE) family, the small multidrug resistance (SMR) family, and the resistance-nodulation‐ division (RND) superfamily [47]. Efflux transporters can be further classified into single or multicomponent pumps. Tetracycline and macrolide transporters are single component ef‐ flux systems that have narrow substrate profiles, while the RND family members have broader substrates and can pump out multiple structurally unrelated compounds [24; 46]. Efflux pumps exist in both Gram-positive and Gram-negative bacteria [48; 49]. MexAB-OprM efflux pumps in Pseudomonas aeruginosa, which belong to RND family, result in higher inhibitory concentration of a large number of antibiotics, such as penicillins, broad‐ spectrumcephalosporins, chloramphenicol, fluoroquinolones, macrolides, novobiocin, sulfo‐ namides, tetracycline and trimethoprim, dyes and detergents [50; 51].

*OM permeability* The OM is an asymmetric bilayer: the phospholipid form the inner leaflet and the lipopolysaccharides (LPS) form the outer leaflet. OM of Gram-negative bacteria pro‐ vides a formidable barrier that must be overcome by drugs. Drug molecules pass the OM by diffusion through porins or the bilayer, or by self-promoted uptake [85]. Small hydrophilic drugs (e.g., β-lactams), enter to the intracellular through the pore-forming porins, while macrolides and other hydrophobic drugs diffuse during their entry [52]. Some resistant clin‐ ical strains of Neisseria meningitidis, K. pneumoniae and Enterobacteraerogenes exhibit a noticeable porin variability resulting in decrease of antibacterial uptake [53]. Reduction of LPS in the outer membrane of Polymyxin-resistant P. aeruginosa strains associates with re‐ sistance development [54].
