Penicillin-Bindi ng Proteins andBacterial Resistance to,3-Lactams

3-Lactam antibiotics are potent, broad-spectrum, bactericidal agents of low toxicity to eukaryotes and widespread clinical use. They encompass an enormous number of mostly semisynthetic compounds which can be conveniently divided into the bicyclic penicillins (penams, penems, carbapenems, oxapenams) and cephalosporins (cephems, cephamycins, oxacephems, carbacephems) and the monocyclic monobactams (39, 109, 110). "Natural" susceptibility to ,-lactams varies widely among bacterial species. For example, Neisseria gonorrhoeae is susceptible to penicillin G at 0.05 ,ug/ml, and thus, a strain for which the MIC was 0.5 ,ug/ml would be considered resistant, whereas a Pseudomonas aeruginosa strain for which the MIC was the same would be considered susceptible (157). Susceptibility to 3-lactam antibiotics reflects the combined effects of binding to targets (penicillin-binding proteins [PBPs]), stability to ,-lactamases, and, in gram-negative bacteria, outer-membrane permeability. Similarly, resistance reflects a change in any of the three components. Bacterial resistance to P-lactams has generally increased in the past 50 years, reflecting the extensive usage of this class of compounds. This minireview focuses on P-lactam resistance mediated by altered PBPs in clinically important bacteria. Traditionally, the major mechanism of 3-lactam resistance has involved P-lactamases, particularly plasmid-mediated 1-lactamases (78, 121). In gram-negative bacteria, P-lactamases are periplasmic and act in combination with altered outer membrane permeability (112, 171). In gram-positive bacteria, they are exocellular, although they are probably associated with the cell wall through electrostatic interactions (138, 140). This topic will not be further discussed here, because there are several excellent reviews on ,-lactamases (2, 4, 14, 15, 78, 121, 139), the interplay between j-lactamases and outer membrane permeability (111-113), and P-lactamase inhibitors (16, 22, 23, 28, 87, 128). There are also several recent reviews on the biochemistry and genetics of PBPs and penicillin-sensitive enzymes (42, 43, 47, 54, 56, 149, 161, 172), topics important for the understanding of PBP-mediated resistance but discussed only briefly in this minireview. For a proper perspective, PBP-associated resistance should be viewed in the broad context of antibacterial resistance (13, 77, 106). The targets for P-lactam antibiotics are cell wall-synthesizing enzymes (penicillin-sensitive enzymes), which are commonly detected by their ability to bind covalently radiolabeled penicillin (hence the name PBPs) (8, 54, 172). Key to their detection is the stability of the penicillin-enzyme complex, which permits analysis by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography (147). PBPs are present in almost all bacteria, but they vary from species to species in number, size, amount, and affinity for 1-lactam antibiotics, usually following taxonomic lines (50). They are localized nonrandomly on the outer face of the cytoplasmic membrane (79) and are anchored through short hydrophobic carboxyor amino-terminal sequences; in gram-negative bacteria, they are pseudoperiplasmic (119). The major enzymatic activities associated with PBPs are peptidoglycan transpeptidase, which is believed to be essential, and DD-carboxypeptidase, which is believed to be dispensable. To date, only PBPs with DD-carboxypeptidase activity have been examined in any great detail. Some PBPs, particularly those of low molecular weight, also have weak P-lactamase activity which, although of no major physiological significance, may nevertheless interfere with PBP binding studies in intact bacteria (93). A given organism contains four to eight PBPs with molecular sizes of 35 to 120 kDa; by convention, these are numbered in the order of decreasing molecular size. Once the number of PBPs for an organism is established, additional PBPs are numbered as derivatives of established ones. This is done to avoid renumbering PBPs and thereby causing confusion with the older literature. For example, when the 91-kDa PBP 1 of Escherichia coli was resolved into two components, they became PBPs la and lb (152), while a novel 78-kDa PBP found in methicillin-resistant Staphylococcus aureus (MRSA) became PBP 2a (66, 134) or PBP 2' (167, 169). Essential PBPs usually have a high molecular mass (60 to 120 kDa), have peptidoglycan transpeptidase activity, and control such fundamental processes as cell growth and division. In a given organism, there are two to four essential PBPs and, thus, potentially multiple ,B-lactam targets. Their inhibition can lead to cell lysis, death, or growth arrest. Binding of P-lactam antibiotics to PBPs is usually measured indirectly, as decreased binding of commercially available radiolabeled penicillin G (50, 89, 152). Altered PBPs associated with P-lactam resistance are more commonly found in gram-positive than in gram-negative bacteria (Table 1). In laboratory strains, high-level, PBP-mediated resistance occurs in several steps rather than as a single-step mutation (148). Possibly, the steric similarity of P-lactams to the natural substrate makes difficult decreased affinity for the former with full retention of catalytic activity toward the latter by the target enzymes. By inference, clinical resistance may require the introduction of multiple amino acid substitutions in the target enzyme(s), in effect, resculpturing the active site (68, 69). Nevertheless, once present in a strain, resistance may rapidly spread along with the resistant strain (clonal spread).