1. Introduction

Bacteria as microbial pathogens are causative agents of life-threatening infectious diseases ^[1]. Such pathogenic bacteria produce alarming numbers in terms of morbidity and mortality outcomes ^[2][3]. One crucial avenue towards bacterial pathogenesis involves the reduction in the therapeutic effects of antibacterial chemotherapy ^[4][5]. Throughout their evolutionary history, bacterial pathogens have developed various means of resisting the inhibitory and bactericidal consequences of antimicrobial agents ^[4]. Such antimicrobial resistance systems involve the engagement of bacterial molecular and cellular-based machinery ^[6]. Interestingly, the selection of a bacterial variant with resistance to a single antimicrobial agent frequently manifests the emergence of a multidrug resistance characteristic in the new mutant ^[7]. Newly emerged bacterial pathogens with resistance to multiple antibacterial agents can result in compromised efficacy in the treatment of infection ^[3][8].

Mechanisms of antimicrobial resistance include the active export systems within the membranes of bacteria, prevention of antimicrobial entrance into cells of pathogenic bacteria, enzymatic destruction of antimicrobial agents, production of thick biofilms, modified targets of antimicrobials, and bacterial sites of action that are protected from antimicrobials, (Figure 1) ^[2][4]. Furthermore, multidrug-resistant bacteria have developed mechanisms that confer the DNA transfer of genetic determinants of resistance to pathogenic species in the clinical setting, the food production industry, the human gut, and in agriculture ^[9].

Figure 1. Bacterial mechanisms of resistance to antimicrobial agents. The common mechanisms of antibiotic resistance in bacteria are enzymatic hydrolysis (1), enzymatic modifications of antibiotics by group transfer and redox process (2), modifications of antibiotic targets (3), reduced permeability to antibiotics by modifications of porins (4), and active extrusion of antibiotics by membrane efflux pumps (5).

Thus, new strategies for the circumvention of bacterial resistance to antimicrobial agents are desired ^[10]. In order to discover novel approaches to address multiple antimicrobial resistances in these microbial pathogens, however, it is necessary to attain a clear understanding of these resistance systems at the molecular and cellular levels. For young and new investigators, here, we consider an introductory overview of each of these disparate bacterial resistance mechanisms here.

2. Enzyme-Based Antimicrobial-Inactivation Systems

Along the timeline of antibiotic discovery and introduction, several enzymatic mechanisms of antibiotic inactivation were also discovered. Although very few novel mechanisms of antibiotic resistance have been reported in recent times, several new variants of known enzymes that endow bacteria with resistance to newly introduced drugs have emerged, suggesting that the bacterial response to new antibiotics or the modified versions of existing antibiotics is swift. The enzymatic mechanisms of antibiotic resistance include hydrolysis, group transfer, and redox processes ^[4]. In terms of diversity, evolution, and spread, antibiotic resistance enzymes contribute remarkably to the bacterial ability to overcome antibiotic pressure. The β-lactamases are the oldest known and the most diverse antibiotic degrading enzymes that cleave the β-lactam ring of the penicillin group of antibiotics and render them ineffective. The first such β-lactamase was discovered soon after the first antibiotic penicillin was in clinical use. Scientific evidence suggests the existence of β-lactamases before penicillin was clinically employed, emphasizing that the production of antimicrobial compounds and the mechanisms to endure them occur in parallel in the environment ^[11]. Bacteria that produce antibiotics apparently require mechanisms to overcome the lethal effects of the compounds, and these are in the form of concurrent production of degradative enzymes, mutations in targets of antibiotics, or active extrusion of antibiotics from the cell so that the antibiotic-producing cell is protected. However, the selection pressure created due to the extensive use of antibiotics in humans and animals propagated the resistant clones of bacteria in clinical and food production environments. In due course of time, genetic exchange mechanisms facilitated the wider dissemination of resistance traits in bacterial communities. The introduction of more antibiotics, newer as well modified, augmented the process of evolution and spread of resistance mechanisms. Since the majority of the antibiotics introduced in the last two decades are mostly the modified versions of existing antibiotics belonging to the same classes (e.g., β-lactams), a few mutations in the enzymes could render bacteria quickly resistant to them ^[11].

The β-lactams constitute the largest group of clinically used antibiotics, comprising of penicillins, cephalosporins of different generations, monobactams, and carbapenems, all of which are characterized by the presence of 3-carbon, 1-nitrogen containing β-lactam ring. The β-lactam antibiotics inhibit the bacterial proteins known as penicillin-binding proteins (PBPs), which perform the critical role of peptide cross-linking during peptidoglycan cell wall biosynthesis. The structural mimicry of the d-Ala-d-Ala terminal fragment of cross-linking peptide by β-lactams facilitates competitive inhibition of PBPs ^[12], which stops the cell wall synthesis leading to bacterial cell lysis and death ^[13]. However, bacteria gain resistance to lactam antibiotics by modifying their PBPs, which are no longer susceptible to binding by the antibiotic. Alternatively, bacteria produce powerful lactamases that degrade antibiotics before they can bind with the PBPs. Since their discovery in the early 1940s, the family of β-lactamases has grown seamlessly, with more than 300 enzymes identified globally ^[14][15].

The early β-lactamases were penicillinase enzymes that degraded penicillin, which started appearing rapidly in clinical bacteria ^[16][17]. The introduction of modified, semisynthetic penicillins such as methicillin, ampicillin, and amoxicillin resulted in the gradual appearance of β-lactamases capable of degrading them. The first plasmid-borne transferrable β-lactamase was TEM-1, followed by TEM-2 and SHV-1 enzymes ^[18][19]. TEM is the most common mechanism of ampicillin resistance compared to less prevalent SHV-1, although both have the same affinity for this antibiotic. TEM and SHV share 60% amino acid similarity between them and are inhibited by clavulanic acid, tazobactam, and sulbactam. The discovery of cephalosporin C in the early 1960s heralded an era of synthetic cephalosporins, which was thought to fend off β-lactamases. Structurally, cephalosporins have their β-lactam ring fused to a six-membered dihydrothiazine ring compared to penicillins in which the β-lactam is fused with a five-membered thiazolidine ring ^[20]. Subsequently, carbapenem and monobactam groups of β-lactam antibiotics with structurally variant lactam rings were discovered from natural sources and formed the basis for the synthesis of similar compounds with modifications. However, the enzymes extended-spectrum β-lactamases (ESBLs) that could hydrolyze a wide range of cephalosporins emerged from TEM and SHV lactamases by point mutations ^[18]. ESBLs hydrolyze a broad spectrum of cephalosporins, including first, second, third-generation cephalosporins and aztreonam, but not cephamycins and carbapenems, and are inhibited by clavulanic acid ^[18][21]. As a consequence of mutations and the expansion of the substrate range, ESBLs have a lesser affinity for classical β-lactams compared to their ancestral β-lactamases. Subsequently, CTX-M type ESBLs with high affinity for cefotaxime emerged independent of TEM and SHV lactamases, and these supposedly evolved from β-lactamases of Kluyvera spp. ^[22]. Over the years, CTX-M has overtaken other ESBLs in terms of number and global distribution, with more than 230 types identified to date. Figure 2 shows the timeline of the evolution of β-lactamases in relation to the introduction of β-lactam antibiotics for clinical use.

Figure 2. Evolution of β-lactamases. Within five decades of discovering the first penicillin-degrading enzyme, β-lactamases capable of hydrolyzing most β-lactam antibiotics, and resistance to inhibitors have emerged. The ability to tolerate a broad spectrum of β-lactams and inhibitor combinations is bolstered by the presence of multiple β-lactamase-encoding genes in a single pathogen.

The initial efforts to classify β-lactamases were based on their functional characteristics such as the substrate-inhibitor profiles, protein molecular weight, isoelectric point, etc. ^[12][14][23]. A second approach employed amino acid sequence similarities and enzymatic activities to classify β-lactamases into four main groups, of which groups A, C, and D are serine β-lactamases, while class B is composed of metallo β-lactamases that require active site zinc ion(s) for their hydrolytic activities ^[12][24]. Group A enzymes form the largest group of lactamases comprising some of the critical resistance enzymes such as TEM, SHV, and CTX-M type of β-lactamases. Other important ESBLs include the carbapenem hydrolyzing KPC type ESBLs originally reported from Klebsiella pneumoniae, which have an expanded substrate spectrum encompassing the cephalosporins and carbapenems but susceptible to inhibition by clavulanates and boronic acid ^[23][25]. The chromosomally encoded AmpC (class C) cephalosporinases described early in the timeline of the discovery of β-lactamases have no homology with penicillinases and thus constitute a distinct group of enzymes ^[26][27]. Commonly found in Enterobacteriaceae, AmpC enzymes are inducible and are produced at low basal levels, and preferentially hydrolyze cephalosporins including cefoxitin but not cefepime. These are generally resistant to inhibition by clavulanic acid, sulbactam, or tazobactam. The metallo-β-lactamases or MBLs belonging to class B have vigorous hydrolytic activities against carbapenems and are also active against a range of cephalosporins ^[28][29]. In 2009, a new variant New Delhi Metallo-β-lactamase (NDM), emerged, and since then, it has been reported from all over the world ^[29]. NDM confers resistance to all β-lactam antibiotics except aztreonam, and the plasmid carrying bla_NDM gene harbors resistance markers for several other antibiotics. VIM and IMP are other important class B carbapenemases commonly encountered in Enterobacteriaceae.

The OXA type enzymes belonging to the Class D lactamase group were originally discovered as plasmid-encoded oxacillin hydrolyzing enzymes in lactose non-fermenting bacteria such as Pseudomonas, Acinetobacter, and Shewanella, and later in Enterobacteriaceae through plasmid exchange ^[30][31]. These enzymes are poorly inhibited by lactamase inhibitors such as clavulanic acid. Although OXA lactamases have a narrow substrate range composed of penicillins, cloxacillin, and oxacillin, the enzymes evolved to hydrolyze extended-spectrum cephalosporins and carbapenems through point mutations, and these abilities vary among different OXA types ^[28][32].

The β-lactamase mediated antimicrobial resistance is widespread among ESKAPE (Enterococcus, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and E. coli) group of organisms, infections with which are usually associated with a significantly higher economic burden and highest risk of mortalities ^[33][34]. The World Health Organization (WHO) has recognized carbapenem-resistant Enterobacteriaceae (CRE) as a serious global health scourge for which the development of new antimicrobials is critically needed ^[35].

Enzymatic hydrolysis is also a common mechanism of resistance against macrolides, rifampicin, and fosfomycin. Many Enterobacteriaceae members produce plasmid-encoded esterases EreA and EreB that hydrolyze the macrolactone ring of 14- and 15-membered macrolides such as erythromycin A, clarithromycin, and azithromycin ^[36][37]. The structurally altered macrolide antibiotic will no longer be able to bind to its preferred target site in the ribosome ^[38].

Another important mechanism of enzymatic degradation is associated with the manganese ion (Mn²⁺)-dependent, chromosomally-encoded FosX that uses water to cleave the epoxide ring of fosfomycin. Other fosfomycin modifying metalloenzymes include FosA, FosB, and two epoxide kinases FomA and FomB ^[39]. FosA is a Mn²⁺ and K⁺-dependent glutathione-S-transferase, while FosB is a Mg²⁺ thiol-S-transferase. The mechanism involves adding glutathione or thiol groups to the oxirane ring of fosfomycin resulting in an inactive drug ^[40]. FomA and FomB kinases utilize ATP and Mn²⁺ ions to phosphorylate the oxirane ring of fosfomycin ^[39].

Tetracyclines are in use for over 70 years as widely used antibiotics in human and animal medicine ^[41]. Tetracycline is broken down by a monooxygenase enzyme Tet(X), which is oxygen- and FAD-dependent ^[42]. Tet(X) monohydroxylates break down tetracyclines at position 11a, followed by non-enzymatic degradation. Similarly, enzymatic monoxygenation of the naphthyl group of rifamycin antibiotics by monooxygenases (Rox) inactivate them by leading to the linearization of the naphthoquinone or naphthohydroquinone ring ^[43].

Enzymatic modification of antibiotics by the transfer of functional groups, such as acyl, glycosyl, ribosyl, nucleotidyl, phosphoryl, or thiol groups, confers resistance to a range of antibiotics, including aminoglycosides, rifamycins, macrolides, epoxides, and chloramphenicol ^[44]. The aminoglycoside modifying enzymes (AME) responsible for resistance to different aminoglycoside antibiotics include N-acetyltransferases (AAC), O-adenyltransferases (ANT), and O-phosphotransferases (APH). These enzymes catalyze the modification of various hydroxyl or the amino groups of the aminoglycosides resulting in their inability to bind to their 30S ribosomal targets ^[45]. Similarly, in Gram-negative bacteria, a plasmid-encoded ADP-ribosyltransferase (Arr-2) is commonly responsible for rifampin resistance ^[46]. Similarly, chloramphenicol is modified by acetyl-CoA-dependent acetylation of its 3-hydroxyl group by chloramphenicol acetyltransferase (CAT) enzymes ^[47]. The modified antibiotic does not bind to its target site, the 50S subunit of ribosomes. CATs are widely distributed among Gram-positive and -negative bacteria and show little amino acid sequence similarities, with only 25 amino acid residues conserved among all CAT variants ^[47].

3. Alteration of Antimicrobial Targets

As bacterial enzymes mentioned above alter drug structures, the drug targets may likewise be altered, preventing drug binding and, thus, conferring resistance. Antimicrobial targets play vital roles in microbial growth or survival and, thus, serve as potentially useful targets for mitigating infection. In addition, these targets must differ or be completely absent from humans or the animal species being treated with an antimicrobial to allow for a selective mode of action. A classic example of such a target is peptidoglycan. Peptidoglycan is essential to the growth and survival of many bacterial species and has a chemical structure that is not present in the mammalian hosts they infect. This allows for the targeting of enzymes responsible for the synthesis and assembly of peptidoglycan. The function of proteins associated with these target sites makes it non-viable for a bacterium to evolve resistance by removing these proteins. However, mutations that allow for continued functionality while reducing the ability of an antimicrobial agent to bind them at the target site have been a veritable regularity in the arms race between antimicrobial substances and antimicrobial-resistant bacteria. In addition to peptidoglycan, alteration in target sites has been attributed to ribosomes, nucleic acid enzymes, and lipopolysaccharides ^[48].

As discussed previously in this review, peptidoglycan inhibition by glycopeptides involves the binding of the peptidyl-d-alanyl-d-alanine terminus of peptidoglycan precursors. This binding prevents integration via the transglycosylase activity of these precursors into the cell wall ^[49], as shown in Figure 3.

Figure 3. Penicillin and penicillin-binding protein of the bacterial cell wall. (1) The peptidoglycan layer of a bacterial cell wall harbors the repeating moieties of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). The NAM subunits bind short variable peptide chains, usually l-Ala and two distal d-Ala residues. (2) The PBP cross-links the peptide side chain, releasing a free Ala. (3) Upon cross-linking, the PBP dissociates from the cell wall. (4) Penicillin binds the PBP active site, affecting its enzyme activity. (5) The β-lactam ring of penicillin is cleaved during its reaction with PBP. Penicillin stays covalently bound PBP, permanently inhibiting the active site. Altered PBPs, such as PBP2a, are unable to accommodate penicillin-binding, preventing cell wall synthesis inhibition ^[48][49].

PCBs are one mechanism for antimicrobial resistance, but the peptidoglycan precursors themselves can undergo alteration, which reduces the affinity of antimicrobials without the involvement of enzymatic inactivation. Such is the case with Enterococcus faecium and E. faecalis, which have been discussed in the literature as developing resistance by acquiring one of two related gene clusters encoding VanA and VanB ^[50][51]. These gene clusters produce a modified terminus that contains d-alanyl-d-lactate as opposed to d-alanyl-d-alanine ^[50]. This alteration leads to glycopeptides having a much lower binding affinity ^[52]. Thus, these gene clusters, found on transposable elements, have allowed the spread of modified targets in enterococci. Similarly, there are rarer but related gene clusters that have been shown to modify peptidoglycan precursors, such as those encoding VanD ^[53], VanE ^[54], and Van G ^[55].

Ribosomes, serving the vital role of protein synthesis, are common to both prokaryotic and eukaryotic organisms but differ quite vastly from one another in structure, making them another suitable candidate for antimicrobial targeting ^[56]. The 50S ribosomal unit serves as the binding site for macrolide, lincosamide, and streptogramin B ^[57]. Recalcitrance to these specific antimicrobials is known as MLS(B) type resistance ^[57], and it results from a post-transcriptional modification of the 23S rRNA component of the 50S ribosomal subunit that is involved with methylation or dimethylation of key adenine bases in the peptidyl transferase functional domain ^[58]. Mutations in the 23S rRNA, close to the site of methylation have also been associated with resistance to the macrolide group of antibiotics in a range of organisms, such as Helicobacter pylori ^[59] and propionibacteria ^[60]. Macrolide resistance in S. pneumoniae has been attributed to an alteration in the L4 and L22 proteins of the 50S subunit ^[61][62]. Oxazolidinones bind to the 50S subunit but have a more complex set of interactions associated with their mechanism of action ^[63]. The translocation of peptidyl-tRNA from the A site to the P site is hindered by this class of antibiotics, but enterococci have been documented to have an altered the P site through the substitution of U in place of G in the peptidyl transferase region (position 2576) of the 23S rRNA, thus resulting in a lowered binding affinity in the 50S subunit for this class of antibiotics ^[64][65][66]. Mutations more closely associated with the A site have been found in E. coli at positions 2032 and 2447 which confer resistance to the oxazolidinone drug linezolid ^[67].

The 30S ribosomal unit is the target of tetracycline and of aminoglycosides, which function by preventing the decoding of mRNA ^[68]. Mutations of the gene encoding 16S rRNA confer resistance to this class of antimicrobials ^[69]. Suzuki and colleagues discovered that substitutions at positions 1400, 1401, and 1483 led to kanamycin resistance in clinical isolates of Mycobacterium, and further strengthened the claim that these changes led to resistance by identifying their absence in kanamycin-sensitive Mycobacterium isolates ^[70]. Position 1400 was the most common substitution, featuring an A to G change ^[70]. The same A to G substitution at position 1408 led to high resistance against amikacin, kanamycin, gentamicin, tobramycin, and neomycin in clinical isolates of Mycobacterium abscessus ^[71].

Active Efflux Pumps of Antimicrobial Agents

Bacastes where intact antiria as microbial pathogens are causative agents enterof life-threatening infectious diseases [1]. Such pathogenic bacterial cells and drug targets are freely accessibleproduce alarming numbers in terms of morbidity and mortality outcomes [2,3]. activOne drug efflux systems can come into play. In this section, we will focus on well-studied antimicrobial transpcrucial avenue towards bacterial pathogenesis involves the reduction in the therapeutic effects of antibacterial chemotherapy [4,5]. Throrughouters, as they make good model systems for study and resistance modulation. B their evolutionary history, bacteria that are l pathogenic frequently make use of integral membrane proteins that function as transporters have developed various means of resisting the inhibitory and bactericidal consequences of antimicrobial agents [964]. Such bacterial transport proteins serve to actively ntimicrobial resistance systems involve the engagement of bacterial molecular and cellular-based machinery [6]. Intexport structurally distinctivestingly, the selection of a bacterial variant with resistance to a single antimicrobial agents from the cytoplasm, where drug targets reside, to the extracellular milieu, where their molecular targets are lacking frequently manifests the emergence of a multidrug resistance characteristic in the new mutant [977]. EffNewlux pumps are present in all y emerged bacteria and are integral parts of l pathogens with resistance to multiple antibacterial physiology, being involved in diverse functions such as the expulsion of toxic products of magents can result in compromised efficacy in the treatment of infection [3,8].

Metchabolism, and maintenance of homeostasis. However, antibiotics as incidental substrates of efflux pumps have resulted in them being viewed largely as nisms of antimicrobial resistance include the active export systems within the membranes of bacterial mechanisms, prevention of antimicrobial resistance. In clinically importantentrance into cells of pathogenic bacteria, such as MDR Mycobacterium tuberculosis, enzymatic destruction of antimethicillin-resistant Staphylococcus aureusrobial agents, Klebsiella pneumoniae, a productiond Pseudomonas aeruginosa, e of thick bioflux pumps have critical roles in ensuringilms, modified targets of antimicrobials, and bacterial survival and evolution into resistant strainsites of action that are protected from antimicrobials, (Figure 1) [2,4]. TFurthesermore, multidrug-resistant bacterial multidrug efflux pump systems are energetically driven by ATP hydrolysis, called primary active transport [98], a have developed mechanisms that confer the DNA transfer of genetic determinants of resistance to pathogenic species in the clinical setting, the food production ind bustry electrochemical ion gradients or ion motive force, the human gut, and in agriculture [9].

Figure 1. Bacterial mechanisms of resistance to antimicrobial agents. The common mechanisms of antibiotic resistance in bacteria are enzymatic hydrolysis (1), enzymatic modifications of antibiotics by group transfer and redox process (2), modifications of antibiotic targets (3), reduced permeability to antibiotics by modifications of porins (4), and active extrusion of antibiotics by membrane efflux pumps (5).

Thus, callned secondary active transport [99,100]. Aw strategies for the ctive transport ofrcumvention of bacterial resistance to antimicrobial agents representsare desired [10]. aIn essential resistance mechanism in bacterial pathogens. Aorder to discover novel approaches to address multiple structurally distinct antiantimicrobial resistances in these microbial agents with disparate modes of action are exported to the extrapathogens, however, it is necessary to attain a clear understanding of these resistance systems at the molecular and cellular milieu, their growth inhibitory properties towardslevels. For young and new investigators, here, we consider an introductory overview of each of these disparate bacteria are diminished,l resistance mechanisms here.

Figure 5. Crystal structure of bacterial ABC efflux pump from S. aureus. The top portion of the ABC transporter Sav1866 is depicted in blue and light blue and represents the two TMDs (sometimes called membrane-spanning domains, MSDs) of the protein, while the orange and red colors depict the two NBDs [104]. The model structure was generated using NGL Viewer [106] of the PDB [107] entries 2HYD and 2ONJ, as reported [104,108].

Figure 6. Classes of well-studied bacterial solute transporters. The bacterial outer and inner (cytoplasmic) membranes are shown. Also depicted are the cytoplasmic and periplasmic spaces. P_i denotes phosphate, and Na⁺ and H⁺ denote sodium and proton, respectively. This figure kindly provided courtesy of Ann F. Varela.

As, like sodium ions or protons. For many if not alldiscussed previously in this review, peptidoglycan inhibition by glycopeptides involves the binding of these peptidyl-d-alantimicrobiyl-d-alanine transporters we do not yet have a clear picture of the nature of the structural configuerminus of peptidoglycan precursors. This binding prevents integrations assumed during each via the transglycosylase activity of the specific steps of theirse precursors into the cell wall [49], transport cycles. Ins shown in Figure 3.

Figure 3. Penicillin and penicillin-binding protein of the bacterial cell wall. (1) The peptidoglycan layer of a bacterial cell wall harbors the repeating moieties of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). The NAM subunits bind short variable peptide chains, usually l-Ala and two distal d-Ala residues. (2) The PBP cross-links the peptide side chain, releasing a free Ala. (3) Upon cross-linking, the PBP dissociates from the cell wall. (4) Penicillin binds the PBP active site, affecting its enzyme activity. (5) The β-lactam ring of penicillin is cleaved during its reaction with PBP. Penicillin stays covalently bound PBP, permanently inhibiting the active site. Altered PBPs, such as PBP2a, are unable to accommodate penicillin-binding, preventing cell wall synthesis inhibition [48,49].

PCBs summary, much work remains to be performed before we can clearly understand the physiologe one mechanism for antimicrobial resistance, but the peptidoglycan precursors themselves can undergo alteration, which reduces the affinity of antimicrobial transport both at fundamental and applied levels of investigs without the involvement of enzymatic inactivation.

Future Directions

8. Concluding Remarks