Chapter 46 Bacterial Cell Wall Synthesis Inhibitors

Abbreviations
CSF	Cerebrospinal fluid
ESBLs	Extended-spectrum β-lactamases
GI	Gastrointestinal
IM	Intramuscular
IV	Intravenous
MIC	Minimal inhibitory concentration
MRSA	Methicillin-resistant Staphylococcus aureus
NAG	N-acetylglucosamine
NAM	N-acetylmuramate
PBPs	Penicillin-binding proteins

Therapeutic Overview

The β-lactam and glycopeptide antibiotics act by inhibiting the synthesis of bacterial cell walls. The β-lactams encompass the widely used penicillins and cephalosporins as well as the carbapenems and monobactams. Although penicillin was first discovered in 1928, it was not until the early 1940s that it was developed as a therapeutic drug. The remarkable results achieved with penicillin therapy revolutionized the treatment of infectious diseases.

Since its introduction, however, many strains of bacteria, particularly Staphylococcus aureus, have become resistant to penicillin through several mechanisms including the production of metabolic enzymes called β-lactamases, altered penicillin binding proteins (PBPs), and reduced drug permeability by resistant bacteria (see Table 45-6). Resistance to penicillin by the induction of β-lactamases led to the development of semisynthetic penicillins resistant to hydrolysis by these enzymes as well as numerous compounds with greater activity against gram-negative organisms. The development of resistance to β-lactams is an ongoing clinical problem and is increasing at a dramatic rate.

β-Lactams are bactericidal under most conditions. Because they inhibit cell wall production, they have maximal activity against rapidly dividing bacteria. The clinically used β-lactams differ in the following ways:

• The organisms against which they are effective

• Their pharmacokinetics, stability, and modes of administration

• The type and extent of resistance found in specific bacteria

In addition to the β-lactams, the glycopeptide drug vancomycin and the topical agent bacitracin are also bactericidal cell wall inhibitors but do not contain a β-lactam nucleus. Vancomycin was isolated originally from an actinomycete in soil. It is active primarily against gram-positive bacteria and came into prominence for several reasons, including the occurrence of methicillin-resistant Staphylococcus aureus (MRSA), the presence of pseudomembranous colitis caused by Clostridium difficile, and the increasing number of organisms resistant to

β-lactams. Resistance to vancomycin, however, is an increasing problem in enterococcus species. Even more alarming has been the discovery of clinical isolates of S. aureus with reduced vancomycin susceptibility, or even full resistance mediated by transfer of resistance genes from enterococcus species.

Therapeutic considerations for the bacterial cell wall synthesis inhibitors are summarized in the Therapeutic Overview Box.

Therapeutic Overview
β-Lactams
Penicillins, Cephalosporins, Carbapenems, and Monobactams
Bactericidal: inhibit many gram-positive and gram-negative organisms
Agents differ by:
Organism inhibited
Pharmacokinetics
Bacterial resistance
Glycopeptides and Polypeptides
Vancomycin—Bactericidal; inhibits many methicillin-resistant staphylococci
Bacitracin—Bactericidal; topical use only for gram-positive bacteria

Mechanisms of Action

β-Lactams

All β-lactam antibiotics have a four-membered ring structure containing a cyclic amide (the lactam); the β indicates that the amine is located on the second carbon relative to the carbonyl group (Fig. 46-1, A). This small ring is structurally strained with low inherent stability, which explains why some penicillins are not effective when administered orally, because they readily undergo hydrolysis, particularly in the presence of high stomach acidity. In addition, this ring is subject to hydrolysis by the β-lactamases, representing a major mechanism of resistance to these compounds. This is why some penicillin derivatives are marketed in combination with β-lactamase inhibitors such as clavulanate, sulbactam, and tazobactam, all of which contain the β-lactam ring structure (Fig. 46-2).

FIGURE 46–1 A. β-Lactam ring structure; the arrow points to the bond that is broken during β-lactamase-catalyzed hydrolysis. B. General structures of the four main classes of β-lactam antibiotics. Variations occur in side-chain (R group) substitutions.

FIGURE 46–2 Structures of β-lactamase inhibitors; note the presence of the β-lactam ring structure.

All of the β-lactam antibiotics, except the monobactams, have a second ring fused to the β-lactam ring (see Fig. 46-1, B). For penicillins the second ring is a thiazolidine, whereas for cephalosporins it is a dihydrothiazine. Carbapenems have an unsaturated ring with an external sulfur. Different structural groups positioned at the side chains (R) give rise to compounds with differing antibiotic properties.

β-lactams interfere with bacterial cell wall synthesis. Although the outer cellular coverings of gram-positive and negative bacteria differ, both have a rigid cell wall composed of a highly cross-linked peptidoglycan matrix (Fig. 46-3). Gram-negative bacteria contain an outer lipopolysaccharide membrane exterior to several peptidoglycan layers. Gram-positive bacteria lack the lipopolysaccharide layer but contain many more (15 to 30) layers of peptidoglycan. The cell wall is assembled in a series of steps, originating within the cytoplasm of the bacteria and terminating outside the cytoplasmic membrane.

FIGURE 46–3 Outer coating of gram-positive and gram-negative bacteria. Gram-positive bacteria have a thicker cell wall composed of many (15 to 30) strands rigid peptidoglycan strands, whereas gram-negative bacteria have a thinner wall (3 to 5 strands) and an added outer membrane. β-Lactam antimicrobials act by inhibiting the synthesis of the rigid peptidoglycan part of the cell wall.

The glycan part of the peptidoglycan is composed of repeating disaccharide units of N-acetylmuramate (NAM) attached to a pentapeptide and N-acetylglucosamine (NAG), connected through β-1,4-linkages (Fig. 46-4). This initial stage of peptidoglycan synthesis occurs in the cytoplasm. The NAG-NAM-pentapeptide is then transferred by a carrier across the cytoplasmic membrane, and the saccharide units are linked in sequence via the pentapeptides to form long chains of alternating disaccharides. This final stage involves a cross-linking reaction to form continuous two-dimensional sheets, a process that occurs outside the cytoplasmic membrane in the periplasm but is catalyzed by membrane-bound transpeptidase enzymes (Fig. 46-5). During the cross-linking reaction, the D-ala-D-ala terminus of the pentapeptide reacts with the transpeptidase to displace the final D-ala, forming an acylenzyme intermediate. This intermediate is reactive and readily couples to the free amino group of the third residue (L-lys) of the pentapeptide of an adjacent chain, thus completing the cross-linking and regenerating the enzyme. It is at the final cross-linking step during synthesis of the rigid peptidoglycan matrix that β-lactam and glycopeptide antibiotics exert their actions, albeit by different mechanisms.

FIGURE 46–4 Repeating glycan portion of the peptidoglycan matrix consisting of the disaccharide N-acetylmuramate (NAM) linked via a lactyl to the pentapeptide and connected to N-acetylglucosamine (NAG) through a β-1, 4-link.

FIGURE 46–5 Polymerization and cross-linking reactions of peptidoglycan stands. The glycopeptide polymer is formed by: (1) polymerization of NAM-NAG complexes via transglycosylation, and (2) cross-linking chains via transpeptidation. Vancomycin inhibits polymerization by binding to the terminal D-ala-D-ala to block linkage of the complex to the elongating glycopeptide polymer, while β-lactams prevent cross-linking by inhibiting the transpeptidase reaction between glycopeptide polymer chains; β-lactams displace the terminal D-ala of the adjacent chain.

Molecular modeling has demonstrated that penicillins and cephalosporins can assume a conformation very similar to that of the D-ala-D-ala peptide, with the reactive β-lactam ring in the same position as the transpeptidase acylation site. Therefore β-lactams undergo acylation, with the β-lactam ring forming a covalent bond with the transpeptidase, inactivating it, and preventing cross-linking.

The multiple β-lactam-sensitive transpeptidases in bacterial cytoplasmic membranes are called PBPs because they covalently bind radiolabeled penicillin G. The PBPs in a given organism are numbered in order by decreasing molecular weight. PBPs of gram-negative and gram-positive bacteria differ; there are usually five PBPs in gram-positive and six in gram-negative organisms. However, a particular numerical designation is not the same protein in different organisms. In addition to their transpeptidase activity, some PBPs show carboxypeptidase or endopeptidase activity and hydrolyze β-lactams.

The effects of exposure to β-lactams depend on the bacterial species and the PBPs to which the drug binds. Some bacteria swell rapidly and burst, some develop into long filamentous structures that do not divide but eventually fragment, with disruption of the organism, whereas others show no morphological change but cease to be viable. Lysis of gram-positive bacteria treated with β-lactams is ultimately dependent on autolysins, which are normally involved in new cell wall synthesis when cells divide. There are bacteria that lack these autolysins, which are termed tolerant because the β-lactams inhibit their growth and division but do not kill them. Thus, in these organisms the β-lactams are bacteriostatic, rather than bactericidal.

Mechanisms of Resistance to β-Lactams

As discussed in Chapter 45, bacterial resistance to antibiotics is a major therapeutic concern. Resistance to the β-lactams is common and occurs by three major mechanisms:

• Destruction by β-lactamase enzymes

• Failure to reach the target PBPs

• Failure to bind to the target PBPs

These mechanisms may coexist, and in some cases it takes a combination of mechanisms, such as decreased permeability and poor binding within a single organism, to confer resistance.

The major forms of resistance of gram-positive bacteria to β-lactams are a consequence of β-lactamases or altered PBPs. Within 2 decades of the first widespread use of penicillin G, most S. aureus showed resistance to the drug. Currently, more than 95% of staphylococci are resistant to penicillin G and ampicillin. These resistances occur by spread of plasmid-encoded β-lactamases, which acylate and cleave the β-lactam ring to inactivate the drug. In gram-positive species such as staphylococci, β-lactamase expression is induced by penicillin, and the enzymes are secreted as exoenzymes.

The presence of PBPs that bind poorly to β-lactams, either due to intrinsic structural features or acquisition of alterations by mutation, also results in resistance to β-lactams, especially in gram-positive organisms. An important clinical example is MRSA, which poses a serious hospital and, increasingly, a community problem. These staphylococci are not inhibited by any currently available β-lactams because of acquisition of a high molecular weight PBP-2a with poor affinity for all β-lactams. The recent increase in penicillin resistance in S. pneumoniae stems from multiple changes in the structure of several PBPs. Several of these changes in more-resistant strains are associated with non-pneumococcal deoxyribonucleic acid inserted into PBP genes by homologous recombination. Enterococci are intrinsically resistant to cephalosporins, because they do not bind to enterococcal PBPs. Aztreonam also fails to bind to the PBPs of gram-positive species and does not inhibit them.

Common forms of resistance in gram-negative bacteria stem from the presence of β-lactamases and failure of the drug to reach the PBPs adjacent to the outer lipopolysaccharide membrane. The β-lactamases of gram-negative bacteria can be encoded by chromosomal or plasmid genes. A wider variety of β-lactamases are produced by gram-negative than gram-positive bacteria.

More than 300 β-lactamases have been described. Mutations that occurred in preexisting β-lactamases after the introduction of new β-lactams have produced the so-called extended-spectrum β-lactamases (ESBLs), which are plasmid-encoded enzymes found mainly in Klebsiella spp. and Escherichia coli. The ESBLs confer high-level resistance to ceftazidime and aztreonam and reduced susceptibility to other third-generation cephalosporins. There are several classification systems for β-lactamases based on structure, spectra of activity, and susceptibility to inhibitors. The β-lactamase inhibitors block the activity of most plasmid-mediated β-lactamases and some chromosomal β-lactamases. However, they do not inhibit the inducible ampC chromosomal β-lactamases expressed by many nosocomial gram-negative pathogens, particularly Enterobacter cloacae and Pseudomonas aeruginosa.

In gram-negative bacteria, β-lactams must pass an outer lipid membrane to reach the PBPs on the cytoplasmic membrane (see Fig. 46-3). Channels in the outer membrane, referred to as porins, allow β-lactams to pass through. Alterations in porin proteins that reduce the amount of drug reaching the PBPs have been observed. For example, P. aeruginosa can delete the porin protein through which imipenem passes and develop resistance. Some β-lactams are extremely resistant to β-lactamases but do not readily pass through porins of gram-negative outer membranes and thus fail to inhibit these bacteria.

Vancomycin and Bacitracin

Vancomycin and bacitracin are two cell wall synthesis inhibitors that are structurally different from the β-lactam compounds and function by different mechanisms. Vancomycin is a glycopeptide with a high molecular weight. It binds to the free carboxyl end of the pentapeptide, sterically interfering with cross-linking of the peptidoglycan backbone. The specificity of the interaction of vancomycin with D-ala-D-ala partially explains the minimal resistance that has been observed with this antibiotic.

However, more than 25% of nosocomial enterococcal isolates, primarily E. faecium, are now vancomycin resistant. Resistance is caused by production of a new pentapeptide ending in a terminal D-ala-D-lac instead of D-ala-D-ala, which does not bind vancomycin. There are also other types of resistance to vancomycin (Table 46-1); the vanA cluster of genes, transferred by a transposable genetic element, is the best characterized. This is an elegant resistance mechanism that contains at least eight genes. One gene product, vanS, functions like a transmembrane receptor, senses the presence of vancomycin, and activates the gene vanR to up regulate expression of three additional genes. The products of these three genes cleave the terminal D-ala-D-ala and insert D-ala-D-lac. Similar resistance genes are present in streptomyces that produce glycopeptide antibiotics and probably evolved as a self-preservation strategy for the organism. Vancomycin-resistant strains of S. aureus are emerging, mediated by increased numbers of vancomycin-binding sites in the cell wall. In 2002, the first two clinical strains of S. aureus isolates with high levels of vancomycin resistance appeared. These strains acquired the vanA gene cluster from vancomycin-resistant enterococci. Bacitracin is a polypeptide bactericidal antibiotic. It inhibits bacterial cell wall synthesis by interfering with dephosphorylation of the lipid carrier that moves the early cell wall components through the membrane.

TABLE 46–1 Resistance of Gram-Positive Bacteria to Vancomycin