Table 1 depicts an expanded version of the functional classification scheme proposed initially by Bush in 1989 (13) and expanded in 1995 (16). This table aligns structural and functional classifications as closely as possible, based on the available information in the public domain. New functional subgroups have been added to the scheme as a result of identification and expansion of major β-lactamase families in which variants continue to be identified on a regular basis (Table 2). As in the earlier functional classifications, enzymes were aligned based on their ability to hydrolyze specific β-lactam classes and on the inactivation properties of the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. A description of each of the functional groups follows.
Group 1 cephalosporinases.Group 1 enzymes are cephalosporinases belonging to molecular class C that are encoded on the chromosomes of many Enterobacteriaceae and a few other organisms (27). They are more active on cephalosporins than benzylpenicillin and are usually resistant to inhibition by clavulanic acid and active on cephamycins, such as cefoxitin. They have a high affinity for aztreonam (Ki values as low as 1 to 2 nM), in contrast to the class A cephalosporinases (14, 15). A few have unusual properties, such as a lack of activity on cefoxitin (6), inhibition by clavulanate or tazobactam (5, 69), or production of resistance to cefotaxime but not ceftazidime (73). In many organisms, including Citrobacter freundii, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa, AmpC expression is low but inducible on exposure to certain β-lactams, such as amoxicillin, ampicillin, imipenem, and clavulanic acid (17, 27, 34, 67). In other organisms, including Acinetobacter baumannii and Escherichia coli, one or more components of the induction system are missing. When produced in large amounts, especially in a host with reduced β-lactam accumulation, group 1 enzymes can provide resistance to carbapenems, especially ertapenem (11, 28, 51). Plasmid-mediated group 1 enzymes in the CMY, ACT, DHA, FOX, MIR, and other families have been known since 1989 but are currently less common than plasmid-mediated subgroup 2be extended-spectrum β-lactamases (ESBLs) (27).
The new subgroup 1e enzymes are group 1 variants with greater activity against ceftazidime and other oxyimino-β-lactams as a result of amino acid substitutions, insertions, or deletions (44). They have been termed extended-spectrum AmpC (ESAC) β-lactamases and include GC1 in E. cloacae (45) and plasmid-mediated CMY-10 (33), CMY-19 (64), CMY-37 (1), and others (21). An AmpC variant from P. aeruginosa with increased activity against imipenem has also been recently described (57). Clinically significant resistance is most often conferred when the producing organism also has a porin mutation (36).
Group 2 serine β-lactamases.Functional group 2 β-lactamases, including molecular classes A and D, represent the largest group of β-lactamases, due primarily to the increasing identification of ESBLs during the past 20 years (Fig. 1). Subgroup 2a penicillinases represent a small group of β-lactamases with a relatively limited spectrum of hydrolytic activity and are the predominant β-lactamases in Gram-positive cocci, including the staphylococci (30) and occasionally enterococci (74). These enzymes preferentially hydrolyze benzylpenicillin and many penicillin derivatives, with poor hydrolysis of cephalosporins, carbapenems, or monobactams at rates usually ≤10% those for benzylpenicillin or ampicillin. An exception is the facile hydrolysis of nitrocefin by the subgroup 2a enzymes. Subgroup 2a β-lactamases are inhibited by clavulanic acid and tazobactam with 50% inhibitory concentrations (IC50s) of usually <1 μM, assuming at least 5 min of preincubation of enzyme and inhibitor. The majority of these enzymes are chromosomal, although some staphylococcal penicillinases are plasmid encoded. This subgroup, which numbered 20 in 1995, has increased to only 25 in 2009. This may be because a true penicillinase does not cause significant clinical resistance for those β-lactams in predominant current use.
Increase in numbers of group 1, 2, and 3 β-lactamases from 1970 to 2009. Shown are group 1/class C cephalosporinases (black), group 2/class A and class D β-lactamases (blue), and group 3/class B metallo-β-lactamases (red).
Subgroup 2b β-lactamases readily hydrolyze penicillins and early cephalosporins, such as cephaloridine and cephalothin, and are strongly inhibited by clavulanic acid and tazobactam. They include the TEM-1, TEM-2, and SHV-1 enzymes, the most common plasmid-mediated β-lactamases identified in the 1970s and early 1980s (38, 58). Since the 1995 β-lactamase compilation (16), at least 9 TEM and 29 SHV 2b enzymes have been described (G. Jacoby and K. Bush, http://www.lahey.org/Studies/) often in the course of characterizing other β-lactamases in unusually resistant clinical isolates.
Subgroup 2be comprises ESBLs. These broad-spectrum enzymes retain the activity against penicillins and cephalosporins of subgroup 2b β-lactamases and in addition hydrolyze one or more oxyimino-β-lactams, such as cefotaxime, ceftazidime, and aztreonam, at a rate generally >10% that of benzylpenicillin. The first and largest subset of subgroup 2be was derived by amino acid substitutions in TEM-1, TEM-2, and SHV-1 that broadened their substrate spectrum at a cost of lower hydrolyzing activity for benzylpenicillin and cephaloridine (53). TEM and SHV ESBLs have been joined by the functionally similar but more rapidly proliferating CTX-M enzymes that are related to chromosomally determined β-lactamases in species of Kluyvera (8). As the name implies, most (but not all) CTX-M enzymes hydrolyze cefotaxime more readily than ceftazidime. Many hydrolyze cefepime as well. Unlike TEM or SHV ESBLs, CTX-M enzymes are inhibited by tazobactam at least an order of magnitude better than by clavulanic acid (8, 65). Finally, there are less common ESBLs unrelated to TEM, SHV, or CTX-M, including BEL-1, BES-1, SFO-1, TLA-1, TLA-2, and members of the PER and VEB enzyme families. Characteristically, subgroup 2be β-lactamases remain sensitive to inhibition by clavulanic acid, a feature used in their detection by clinical laboratories (19).
Subgroup 2br enzymes are broad-spectrum β-lactamases that have acquired resistance to clavulanic acid (IC50 ≥ 1 μM) and related inhibitors while retaining a subgroup 2b spectrum of activity. Currently 36 of the 135 functionally characterized TEM enzymes have this property and include enzymes such as TEM-30 and TEM-31 (IRT-2 and IRT-1, respectively), as well as 5 of the corresponding functionally characterized 72 SHV enzymes (e.g., SHV-10). No CTX-M β-lactamase demonstrates this characteristic to date (G. Jacoby and K. Bush, http://www.lahey.org/Studies/).
Subgroup 2ber includes TEM enzymes that combine an extended spectrum with relative resistance to clavulanic acid inhibition. Although all have clavulanic acid IC50s greater than that of TEM-1 (0.08 μM), for some 2ber enzymes the increase in clavulanic acid resistance is modest. They have also been termed CMT (complex mutant TEM) β-lactamases and include TEM-50 (CMT-1) (56, 61).
Subgroup 2c penicillinases are characterized functionally by their ability to hydrolyze carbenicillin or ticarcillin at least 60% as rapidly as benzylpenicillin, with cloxacillin or oxacillin hydrolyzed at rates less than half those for benzylpenicillin (16). These penicillinases are generally easily inhibited by clavulanic acid or tazobactam, most often with IC50s of <1 μM. Because carbenicillin is an antibiotic that is currently used infrequently and is not tested for stability by most investigators, only a few new 2c β-lactamases have been described over the past decade (18, 40, 47).
Subgroup 2ce contains the recently described extended-spectrum carbenicillinase RTG-4 (CARB-10) with expanded activity against cefepime and cefpirome (50).
Subgroup 2d includes β-lactamases distinguished by their ability to hydrolyze cloxacillin or oxacillin at a rate of >50% that for benzylpenicillin and hence are known as OXA enzymes. Carbenicillin may also be readily hydrolyzed. Most members of the OXA family, however, are currently identified according to their conserved amino acid motifs rather than according to function. Many β-lactamases in this subgroup are inhibited by NaCl; they typically have clavulanic acid IC50s of ≥1 μM. OXA-related enzymes now comprise the second largest family of β-lactamases (Table 2).
In the new subgroup 2de are cloxacillin- or oxacillin-hydrolyzing enzymes with an extended spectrum that includes oxyimino-β-lactams but not carbapenems. The majority of 2de enzymes are derived from OXA-10 by between 1 and 9 amino acid substitutions and include enzymes such as OXA-11 and OXA-15. They have most often been found in Turkey and France in isolates of P. aeruginosa, where the level of resistance they produce is higher than that in E. coli (9). Resistance to ceftazidime is usually more pronounced than resistance to cefotaxime or aztreonam. However, organisms producing a few oxacillinases, such as OXA-1 or OXA-31, may be susceptible to ceftazidime but resistant to cefepime (4).
New subgroup 2df β-lactamases are OXA enzymes with carbapenem-hydrolyzing activities. They appear most frequently in Acinetobacter baumannii and are usually produced by genes that are located on the chromosome (66), although plasmid-borne OXA-23 and OXA-48 enzymes have been identified in the Enterobacteriaceae (16, 48). The 2df enzymes have been divided into nine clusters according to amino acid homologies (52, 59, 62, 66). Although subgroup 2d enzymes are defined functionally according to their ability to hydrolyze cloxacillin or oxacillin, only a few subgroup 2df enzymes have been tested using these substrates (66). Of those tested, only OXA-50 had no detectable oxacillin hydrolysis. The characterized OXA carbapenemases have weak hydrolytic activity for carbapenems, demonstrated by kcat values for imipenem and meropenem that are generally ≤1 s−1, with imipenem hydrolyzed faster and more efficiently than meropenem. These rates compare to much higher kcat values for benzylpenicillin or oxacillin, substrates that were usually hydrolyzed at least 40- to 50-fold faster than the carbapenems (66). Although the producing organisms are generally highly resistant to carbapenems, E. coli transformants or transconjugants that produce these enzymes are usually susceptible to the carbapenems (66). The enzymes, and their producing organisms, are typically unresponsive to inhibition by clavulanic acid.
Characteristics of the subgroup 2e cephalosporinases include the ability to hydrolyze extended-spectrum cephalosporins and to be inhibited by clavulanic acid or tazobactam. The inducible, chromosomal cephalosporinases in the Proteae often belong to this subgroup. They can be confused with the group 1 AmpC enzymes or with ESBLs because they may appear in similar organisms and with comparable resistance profiles. Subgroup 2e enzymes can be differentiated from AmpC enzymes by their poor affinity for aztreonam, in contrast to the nM Km (Ki) values observed for aztreonam with group 1 enzymes (13). The number of 2e enzymes has remained stable since 1995 and is not expected to include many new members in the future, probably because many of these enzymes are now identified as ESBLs.
Serine carbapenemases from molecular class A populate subgroup 2f. Carbapenems are the distinctive substrates for these enzymes, which can be inhibited better by tazobactam than by clavulanic acid. Extended-spectrum cephalosporins such as ceftazidime are not well hydrolyzed by the SME and IMI-1 enzymes, but aztreonam can be degraded by most of them, except for GES-3 and GES-4. The SME family, as well as IMI-1 and NMC-1 β-lactamases, are representatives of the chromosomal subgroup 2f enzymes (52). More worrisome, however, are the plasmid-encoded subgroup 2f β-lactamases, including KPC and some GES (formerly IBC) enzymes. The KPC carbapenemases in particular have recently been associated with major outbreaks of multidrug-resistant Gram-negative infections in hospitals, including those in the New York City metropolitan area (10, 12, 70) and in Israel (32), with their spread now becoming worldwide (43, 63, 68).
Group 3 MBLs.Metallo-β-lactamases (MBLs), a unique group of β-lactamases both structurally and functionally, are usually produced in combination with a second or third β-lactamase in clinical isolates. They differ structurally from the other β-lactamases by their requirement for a zinc ion at the active site. Functionally, they were once distinguished primarily by their ability to hydrolyze carbapenems, but some serine β-lactamases now have also acquired that ability. In contrast to the serine β-lactamases, the MBLs have poor affinity or hydrolytic capability for monobactams and are not inhibited by clavulanic acid or tazobactam. Instead, they are inhibited by metal ion chelators such as EDTA, dipicolinic acid, or 1,10-o-phenanthroline (31, 37). These metalloenzymes have been subdivided, based on either structure (subclasses B1, B2, and B3) (22-24) or function (subgroups 3a, 3b, and 3c) (54). As with the other functional groups, the two groupings were aligned as closely as possible, although structural subclasses B1 and B3 were found to correlate with similar functions (Table 1). MBLs originally were identified as chromosomal enzymes in Gram-positive or occasional Gram-negative bacilli, such as Bacteroides fragilis (72) or Stenotrophomonas maltophilia (35, 42), and their number accordingly remained relatively constant for many years. When MBLs began to appear on transferable elements, they became more promiscuous and were subject to evolutionary pressures in a variety of hosts, resulting in enzyme families with several dozen unique variants (Table 2).
Based on more extensive biochemical characterization of the increasing numbers of metallo-β-lactamases, it is now being proposed that only two functional subgroups be described. Subgroup 3a includes the major plasmid-encoded MBL families, such as the IMP and VIM enzymes that have appeared globally, most frequently in nonfermentative bacteria but also in Enterobacteriaceae (52). These enzymes belong to molecular subclass B1 based on the consensus amino acids that serve as ligands to the two zinc atoms required for the broad-spectrum hydrolytic activity observed with these MBLs (22-24). In addition, the common L1 MBL from S. maltophilia as well as the subclass B3 MBLs, such as CAU-1, GOB-1, and FEZ-1, are being added to subgroup 3a (7, 54). These enzymes differ from the other subgroup 3a enzymes due to differences in the amino acids involved in zinc binding; however, both structural subclasses require two bound zinc ions for maximal enzymatic activity and have similar broad-spectrum substrate profiles (23, 24). High hydrolysis rates based both on kcat and kcat/Km values are observed for penicillins, cephalosporins, and carbapenems, but not monobactams. An exception is the FEZ-1 carbapenemase with preferential hydrolysis of cephalosporins compared to carbapenems and penicillins, due primarily to high Km values for these latter substrates (41).
Subgroup 3b contains a smaller group of MBLs that preferentially hydrolyze carbapenems in contrast to penicillins and cephalosporins (60). These enzymes have been difficult to detect when chromogenic cephalosporins, such as nitrocefin, are used to monitor the presence of β-lactamase activity on isoelectric focusing gels or during purification procedures. Thus, the chromosomal MBLs in Aeromonas spp. were often overlooked in carbapenem-resistant isolates because the enzymes did not react with nitrocefin in cell extracts used for isoelectric focusing or during chromatography (71). Mechanistically, these enzymes are most effective in hydrolyzing carbapenems if only one of the zinc binding sites is occupied (26). In contrast to the other subgroups of MBLs, the presence of a second zinc ion is actually inhibitory to enzymatic activity (20).
Group 4 β-lactamases previously included in the 1995 functional classification have been omitted in the present scheme. These enzymes most likely would be included in one of the existing enzyme groups if more information about them were available. Because these enzymes have as yet been incompletely characterized, further categorization has not been attempted.
Ingenuity of the β-Lactam as the Acylating Agent
β-Lactams occur relatively rarely in nature. Initial work by Strominger indicated that the activity of penicillin was due to the inherent strain of the four-membered ring or to the reduced amide resonance [1,2]. However, many amide and lactam derivatives are as chemically reactive as the penicillins and cephalosporins [3,4]. This raises the question of what factors account for the seemingly special nature of β-lactam antibiotics as compared to other acylating agents [5,6,7,8]. A shift in the understanding of the biological activity of β-lactams from the traditional view of penicillins as effective acylating agents, to the necessity for a proper molecular recognition between the lactam or amide and its host protein has occurred [3,9]. Apparently, all β-lactams with a current therapeutic application operate by means of mechanisms resulting in the formation of hydrolytically stable enzyme complexes. Derived biochemically from two molecules of l-cysteine, penicillin has been designed by fungi to mimic the d-alanine-d-alanine termini of bacterial peptidoglycans, which allows it to be recognized by transpeptidases. The latter interlinks peptidic residues between peptidoglycan strands in the bacterial cell wall . This crucial step for securing the integrity of the bacterial cell wall is interrupted by penicillin, which irreversibly acylates the active site serine of the transpeptidases. For “classical” bicyclic β-lactam antibiotics, (Figure 1) the antibacterial activity is due to the position of the lactam nitrogen in the ring fusion, which allows for sufficient pyramidalization of the nitrogen center, which, in turn, perturbs the resonance stabilization of the lactam amide. Therefore, the N-fused bicyclic β-lactams have enhanced lactam electrophilicity toward nucleophilic ring opening, easily reacting with nucleophilic amino acid side chain functionalities, including those essential to proper enzyme function. Recently, it has been hypothesized that the acylating ability of the penicillin-like β-lactam antibiotics, approximated to that of acid chlorides [2,11], is aided by an intramolecular protonation of the lactam nitrogen by the neighboring carboxylic acid residue within the active site of the transpeptidases .
Figure 1. Clinically relevant β-lactam antibiotics.
Figure 1. Clinically relevant β-lactam antibiotics.
Based on experimental data obtained from inelastic neutrons and quantum chemical theory, it has been suggested that penicillin changes from relatively inactive at near neutral physiological pH, to a highly electrophilic amide in the acidic active site environment . This shift in reactivity could explain the ability of penicillin to travel to its molecular target, the bacterial cell wall transpeptidases, in the mammalian organism unaltered and demonstrate yet again the cleverly designed weapon at the molecular level—the appropriately substituted β-lactam as the acylating reagent. The hydrolysis rate of unsubstituted 2-azetidinone is considerably slower in acidic media and is virtually unchanged under basic conditions compared to that of the penicillin .
In 1981, two independent groups from the Squibbs and Takeda laboratories reported the isolation of the first N-thiolated β-lactams derived from natural sources. These β-lactams differed from all previously reported antibiotics by having a monocyclic ring with an N-sulfonic acid group attached directly to the nitrogen (Figure 1). They named these “monobactams” to highlight, for the first time, that monocyclic β-lactams are present in the environment and have potent antimicrobial activities. Aztreonam, the first marketed monobactam, has activity against most aerobic Gram-negative bacilli, including Pseudomonas aeruginosa. These reports confirmed that β-lactams do not strictly require a conformationally constrained bicyclic ring structure to possess antibacterial properties. These monobactams display a broad spectrum of activity against aerobic Gram-negative bacteria, but little or no activity against Gram-positive bacteria, such as Staphylococcus aureus.
Recently, Csizmadia and co-workers defined a new “amidicity” index, which is used to quantify the relative amide character for a wide range of amides [13,14,15]. Their method utilizes computed enthalpies of the hydrogenation of the amide carbonyls, which should reflect the degree of amide character. In addition, two independent computational methods have been used by Glover and co-workers for the determination of the amidicity of a range of amides, including β-lactams . In the latter work and elsewhere, monocyclic β-lactams have been computed to be planar at the nitrogen, but bicyclic systems in the penam/em and cepham/em scaffolds have varying degrees of twists about the lactam C−N bond and pyramidalization at the nitrogen, which, with the exception of the cepham system, result in reduced amidicities relative to N,N-dimethylacetamide (the archetypical amide). It is interesting to note that these computational results indicate that the loss of amidicity, even in the highly pyramidal penam and penem scaffolds, is not excessive, which accounts for the stability of β-lactam antibiotics to side reactions in the transport of β-lactam antibiotics to their target enzymes. Moreover, since correlations of reactivity with factors, such as pyramidality and, therefore, amidicity alone, have been determined to be poor, this supports the view that the transport, metabolism and target binding characteristics of β-lactam antibiotics must play the dominant role in their biological activity [3,9].
2. PBPs and β-Lactamases: the Two Main Molecular Targets for Drug Development, So Far
For more than seven decades, penicillins and related antibiotics have been used widely for the control and treatment of bacterial infections [17,18]. As many as 40 structurally different β-lactam compounds in 73 formulations are currently available for medical use. Improving upon the effectiveness of this class of antimicrobial agents has been an ongoing challenge, one which has continued to attract increasing attention, because of the emergence of multidrug-resistant strains of bacteria [19,20,21,22,23,24]. Over the years, countless penicillin derivatives [25,26,27] have been prepared and tested, and a variety of new β-lactam ring systems have been introduced (Figure 1). These systems include the penems, cephalosporins, carbapenems, oxapenams, oxacephams, as well as monocyclic, spirocyclic and multicyclic ring systems.
At least three families of bacterial enzymes specifically recognize β-lactam antibiotics. These include transpeptidase enzymes, or penicillin binding proteins (PBPs), which are the inhibitory targets for antibiotics, β-lactam synthases, the enzymes that biosynthesize penicillin, cephalosporins and monobactams, and β-lactamases, the defense enzymes of many drug-resistant bacteria. β-Lactamases are generally divided into serine- and zinc-dependent enzymes . PBPs and Class A, C and D β-lactamases have an active-site serine, a property they share with a large class of enzymes known as serine proteases. Class B represents the bacterial metallo-β-lactamases.
In 1976, Jean-Marie Frère and coworkers  suggested that an “active site” model of the PBPs can be represented kinetically:where E is the enzyme, S the antibiotic, ES a non-covalent complex, ES* a covalent acyl-enzyme and P(s) the inactivated product(s) of degradation of the antibiotic. Efficient inactivation of the enzyme depends on the rapid and nearly quantitative accumulation of the ES* complex, which is the result both of its stability (a low k3 value) and its rapid formation (generally due to high k2 values).
In subsequent years, it became clear that the above model depicts how β-lactams interact not only with PBPs, but also with a large number of β-lactamases, involving in all cases the initial acylation of the active site serine . Plasmid-mediated production of β-lactamases is largely responsible for the resistance of many bacteria to the normally lethal action of β-lactam antibiotics. There has been much discussion on the evolutionary relationship between β-lactamases and transpeptidases [31,32,33,34,35,36]. Several reviews on the kinship between PBPs and serine β-lactamases [36,37,38], which belong to the superfamily of β-lactam recognizing enzymes [34,39,40,41,42], have appeared in the literature. It is the more rapid deacylation rate of the β-lactamases that separates them from the PBPs. Thus, nature appears to have taken a basic conserved protein template and, through mutation and selection, to have produced two types of bacterial enzymes. These enzymes differ from one another, but both have vital functions for the survival of the bacteria. There is little similarity between the PBPs, the serine β-lactamases and the rest of the serine enzymes, either structurally or with regard to amino acid (AA) sequence [43,44,45,46].
Although reports emerged as early as 1940 that some strains of bacteria can exhibit resistance to penicillin, this had no clinical relevance until the 1970s [47,48]. Today, resistance to antibiotics is a global crisis [49,50,51,52] with multiple drug resistance (MDR) and extreme drug resistance (XDR) reported in both community and healthcare settings . Bacterial resistance to β-lactams generally operates by three different mechanisms: decreased access of antimicrobials to the target PBPs (efflux pumps), altered PBPs (affinity of binding decreased) and β-lactamase production. The latter is by far the most efficient of the resistance mechanisms . Two strategies have been used against the action of the β-lactamases. One is the design of β-lactams that are poorer substrates for many common β-lactamases, including the so-called extended spectrum β-lactamases, whose distribution in nature is expanding. These agents include the third-generation cephalosporins, the carbapenem, imipenem, and the monobactam, aztreonam. The other is the design of β-lactamase inhibitors. The search for β-lactamase inhibitors has led to the identification of clavulanic acid, tazobactam and sulbactam  (Figure 2), which have been introduced into the market in formulations  combining them with a variety of β-lactam antibiotics (inhibitors of PBPs) .
Figure 2. Clinically relevant β-lactamase inhibitors.
Figure 2. Clinically relevant β-lactamase inhibitors.
Currently, the majority of clinically relevant β-lactamase inhibitors have activity against the Class A β-lactamases, the most common class at this time [37,38,58]. Recently, inhibitors such as avibactam, currently in Phase III clinical trials with activity against Class C and D β-lactamases, have been reported . The structures of numerous potential β-lactamase inhibitors have been synthesized and tested in the last few decades [58,59,60,61,62]. The history, microbiology, synthesis and mechanism of the inhibition of the β-lactamases have all been reviewed [5,6,7,8,58,63,64,65,66].
3. β-Lactams Lacking the Ionizable Residue at the Lactam Nitrogen: A New Direction of Antimicrobial Compounds against Bacteria
All of the clinically relevant β-lactam antibiotics and those in clinical trials [67,68] contain an ionizable group either in the proximity (carboxylic acid, bicyclic, penicillin-like structures) or on (sulfonic acid, monobactams) the lactam nitrogen of the β-lactam ring. Until recently, it was generally accepted that for β-lactams to exert bactericidal activity, they must contain a scaffold, which specifically has an ionizable group at the lactam nitrogen within 3.6 Å of the β-lactam carbonyl carbon. However, there now appear to be exceptions to this scaffold requirement, since N-alkylthiolated β-lactams possess inhibitory, although not cidal, antimicrobial activity . Subsequent reports have confirmed that once that ionizable group is “removed” from the lactam nitrogen, a variety of novel molecular targets begin to emerge [70,71,72].
The synthesis and the biological evaluation as antibacterial agents of monocyclic β-lactams with an alkylthio group on the lactam nitrogen [69,73,74,75,76], stable to the hydrolytic activity of β-lactamases (Figure 3), have been reported in the literature in the last decade. The initial studies of N-thiolated β-lactams have focused on the determination of their structure-activity toward Staphylococcus, especially methicillin-resistant S. aureus strains (MRSA). There are two important observations that have come from this work, so far: (1) the ring functionality does have a role, albeit still a rather undefined one, on the in vitro anti-MRSA properties of the β-lactams; and (2) most significantly, the N-organothio moiety is an absolute requirement for bioactivity. To extend these investigations further, the in vitro activity of the more promising compounds against S. aureus have been evaluated for a much wider range of Gram-positive and Gram-negative bacteria, totaling 23 genera, with more than one species or strain. While most of the bacteria tested were not affected by these β-lactams, a few important pathogenic genera, such as Staphylococcus, Micrococcus and Bacillus, were affected [69,73,74,75,76].
Figure 3.N-alkylthio β-lactams active against Staphylococcus spp. (including methicillin-resistant S. aureus (MRSA)) and Bacillus spp.
Figure 3.N-alkylthio β-lactams active against Staphylococcus spp. (including methicillin-resistant S. aureus (MRSA)) and Bacillus spp.
Thus, the selection of bacteria affected by the β-lactams is extremely narrow. The latter are highly selective towards Staphylococcus spp. and Bacillus spp. Moreover, unlike penicillins, which inhibit cell wall crosslinking enzymes, N-thiolated lactams are characterized by a bacteriostatic activity and act through a different mechanism of action . While how these N-thiolated β-lactams exert their microbiological effects is still not completely understood, they clearly affect cellular processes associated with coenzyme A and lipid biosynthesis . Recently, the representatives of these monocyclic N-methylthio-azetidinones were even reported as selective inhibitors of histone deacetylases (HDACs) . In that case, the presence of an N-methylthio group had a key role in providing to the new β-lactams a stringent isoform selectivity. Many staphylococcal infections are associated with the development of resistance to β-lactam antibiotics. This is particularly important in patients with chronic diseases, such as cystic fibrosis (CF), where persistent colonization by pathogenic bacteria occurs and the repeated use of antibacterial agents selects for specific resistant strains. A rise in S. aureus infections has been reported in CF patients, with an increase in the prevalence of highly virulent MRSA . The addition of a polyphenolic moiety to the N-alkylthio β-lactams proved to be of importance for activity in case of CF  (Figure 4).
The presence of oxidative stress in CF due to an increased production of reactive oxygen species (ROS) and to an impaired antioxidant status, particularly during chronic pulmonary infections, points to new therapeutic possibilities in targeting anti-oxidant pathways . Thus, the necessity for antioxidant properties in the structure of an antibacterial agents against CF led to the preparation of dual activity N