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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: IUBMB Life. 2018 Jun 22;70(9):881–888. doi: 10.1002/iub.1875

Have we realized the full potential of β-lactams for treating drug-resistant TB?

Elizabeth Story-Roller 1, Gyanu Lamichhane 1,*
PMCID: PMC6119476  NIHMSID: NIHMS964038  PMID: 29934998

Abstract

β-lactams are the most widely used antibiotics and are effective against a spectrum of pathogenic bacteria. Here, we focus on the state-of-the-art understanding of the molecular underpinnings that determine the overall efficacy of β-lactams against TB and also include historical perspectives of this antibiotic class against this ancient disease. We summarize literature that describes why earlier generations of β-lactams are ineffective and the potential promise of newer β-lactams that exhibit improved efficacy against TB. Emerging evidence warrants renewed consideration of newer β-lactams in regimens for treatment of drug-resistant TB.

β-lactams are the most widely used class of antibiotics to treat bacterial infections in humans (1). They have a long and rich history of usage in clinical medicine in treating a broad range of bacterial infections. Their well-established safety and efficacy profiles, wide availability, and affordability have made them a cornerstone of modern antibacterial treatment regimens. The commercially available β-lactam antibiotics in clinical use today represent five sub-classes, namely, penicillins, cephalosporins, monobactams, carbapenems, and penems; roughly in the chronological order in which they were introduced into the clinic. All drugs within each sub-class share a common four membered β-lactam ring (Figure 1) thought to mimic the peptide bond present in native substrates in bacteria. A sixth sub-class is clavams, which are not known to possess antibacterial activity by themselves, but rather potentiate other β-lactams by inactivating β-lactamases. Each β-lactam sub-class differs in its chemical composition and structure of the cyclic ring fused to the central β-lactam ring. Further, members within each sub-class vary in the composition of the side chains that decorate the bicyclic ring system.

Figure 1.

Figure 1

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Structures of the β-lactam core ring (top) and core bicyclic ring systems in the five major categories of β-lactam antibiotics.

Tuberculosis (TB) was the major infectious disease globally when penicillins were discovered. Penicillins exhibited potent activity against a wide range of bacteria, but lacked therapeutically valuable activity against Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (2). Two potential reasons were presented to explain this conundrum.

β-lactamase

The presence of a robust β-lactamase that was known to inactivate β-lactams (available at the time) was considered to be the primary reason why penicillins and cephalosporins were not potent against Mtb (3). Now we know that this β-lactamase, BlaC, is chromosomally encoded and hydrolyzes penicillins and cephalosporins robustly (4), as well as carbapenems to a limited extent (5). Mtb lacking BlaC exhibits enhanced susceptibility, especially to penicillins (6). A β-lactamase inhibitor such as clavulanate, when used as a companion agent, can reduce the minimum inhibitory concentration (MIC) of penicillins and cephalosporins against Mtb to a large extent (7, 8), but in general it has a lesser effect on the activity of most carbapenems (9). The identification of additional proteins with β-lactamase activity, Rv0406, Rv3677 (10) and CrfA (11), has further complicated the relevance of these proteins in the metabolism of β-lactams. Additional studies will be necessary to unveil the overall relevance of these proteins in the treatment of Mtb infections using β-lactams.

Cell wall as a barrier

Mtb has an unusually thick cell wall composed of complex long chain sugars, fatty acids, and lipid molecules (12, 13). Historically, the unique biochemical makeup of the cell wall has been postulated to limit diffusion of β-lactams into the peptidoglycan where the enzyme targets, D,D-transpeptidases (DDTs), commonly known as penicillin binding proteins, reside. Perhaps initially presented as a hypothesis to account for the intrinsic resistance of Mtb to the β-lactams available at the time, this hypothesis has casually transitioned as an explanation, but without direct experiments to test it. On the contrary, Chambers et al. demonstrated that concentration equilibration of β-lactams across the Mtb cell wall was achieved within several minutes and the rate of penetration was similar to that of many bacteria that were susceptible to penicillins (14). The possibility of existence of robust efflux pumps in the cell wall that can effectively reduce β-lactam concentration, such as one encoded by Rv0194, has also been postulated (15), but their identities or activities have yet to be established with direct evidence.

Clinical utility of β-lactams for treatment of TB

The increasing prevalence of resistance to first-line therapy, in addition to the poor efficacy and safety profile of second-line therapies, highlighted the pressing need for developing new treatment regimens that are effective, well-tolerated, and affordable. For these reasons, there was renewed interest in evaluating the potential of using β-lactams for treatment of drug-resistant TB (DR-TB). In the 1990s, anecdotal reports described the efficacy of regimens containing amoxicillin/clavulanate for treatment of DR-TB (16, 17). Evaluation of early bactericidal activity (EBA) of amoxicillin/clavulanate in TB patients also showed promising activity (18). EBA of a drug is the reduction in bacterial burden that it produces after a few days of treatment. However, subsequent case studies have noted conflicting data. For example, EBA was not observed in a study of amoxicillin/clavulanate in patients with drug-susceptible TB (19). Unlike penicillins and cephalosporins that are susceptible to β-lactamase activity in Mtb, carbapenems are known to be relatively more resistant (5, 20). In the 2000s, evidence of potential in vivo activity of carbapenems came from observations in mouse models of TB, in which imipenem and meropenem showed promising efficacies (21–23). In 2005, Chambers et al. described promising results from a small cohort of patients treated with regimens containing imipenem (21). This study is likely to be the first systematic evaluation of a carbapenem for treatment of TB. In recent years, multiple reports have described use of carbapenems for treatment of DR-TB, some with efficacy as high as 80% in treating extensively drug-resistant TB (XDR-TB) (24–26). In 2016, the first clinical trial to evaluate carbapenems demonstrated a significant EBA of meropenem/clavulanate in TB patients (27). Recent observations of efficacy of carbapenems in treating TB are reviewed elsewhere (28). Below we will describe β-lactam targets in Mtb and discuss literature relevant to why only some β-lactams exhibit activity against TB.

Non-classical peptidoglycan of Mtb and inhibition of its synthesis by β-lactams

β-lactams exert their activity by inhibiting synthesis and metabolism of peptidoglycan, a critical component of the bacterial cell wall. Peptidoglycan is a three-dimensional exoskeleton that encapsulates the plasma membrane and provides bacterial cells their shapes and rigidity. It also serves as an anchor for many molecules that decorate the cell wall. In Mtb, components of the cell wall, such as, arabinogalactans, are covalently linked to the peptidoglycan layer (29).

As can be inferred from the name, peptidoglycan is a polymer of sugar (glycan) molecules and peptides (Figure 2). The glycan chain is comprised of repeats of disaccharide N-acetyl-glucosamine-N-acetyl-muramic acid cis linked by a glycosidic bond generated by transglycosylase. Each N-acetyl-muramic acid moiety is covalently linked to a peptide side chain. The peptide side chain contains a mixture of L- and D-amino acids. For instance, the pentapeptide L-alanyl-D-glutaminyl-meso-diaminopimelyl-D-alanyl-D-alanine and tetrapeptide L-alanyl-D-glutaminyl-meso-diaminopimelyl-D-alanine are the commonly found peptide side chains in the peptidoglycan of Mtb. The final reaction in peptidoglycan synthesis involves linking of two amino acids in adjacent peptide chains with a transpeptide bond generated by transpeptidases.

Figure 2.

Figure 2

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Model of M. tuberculosis peptidoglycan. L-alanine (L-Ala), D-glutamine (D-Gln), meso-diaminopimelic acid (m-DAP) and D-alanine (D-Ala). The hexagonal structures depict sugars N-acetylglucosamine (white) and N-acetylmuramic acid (blue).

The model that describes the biosynthesis and structure of peptidoglycan is predominantly influenced by observations from model organisms such as E. coli and B. subtilis. Based on this historical model, the existence of only one class of transpeptidases, namely the DDTs, was known and crosslinking of the stem peptides was considered to be catalyzed primarily by this enzyme class or its related variants (29). DDTs covalently link the fourth amino acid of one peptide to the third amino acid of the adjacent peptide and form a 4→3 transpeptide bond. In 1974, it was reported that the majority of the stem peptides in Mtb peptidoglycan were linked between the third amino acid of one stem peptide to the third amino acid of another with a 3→3 transpeptide bond (30). This observation was ignored for a long time, as the enzymes that would generate these non-classical transpeptide bonds were not known or could not be accounted for within the historical model of peptidoglycan. The enzymes responsible for these 3→3 transpeptide bonds in Mtb were identified (31–33) following their discovery in Enterococcus faecium (34) and are referred to as L,D-transpeptidases (LDTs). Today, we know that the final step of PG synthesis in Mtb requires two enzyme sets, LDTs and DDTs, that generate 3→3 and 4→3 linkages, respectively.

The precursors for building peptidoglycan in Mtb are disaccharyl-tetrapeptide or disaccharyl-pentapeptide. The dipeptide in the free end of the peptide chain is bound by transpeptidases as a substrate to generate the transpeptide linkages. While LDTs use tetrapeptide substrates Ala to generate 3→3 transpeptide linkages (35), DDTs use pentapeptide substrates to generate 4→3 linkages (29). Biosynthesis of Mtb peptidoglycan is reviewed in more detail by Pavelka et al (36).

Mechanisms of β-lactam activity against Mtb

β-lactams are known to mimic D-alanyl-D-alanine, or the structure and the chemical composition of the free end of the peptide side chains before they are crosslinked by transpeptidases (37). By mimicking the native substrates, β-lactams undergo an initial acylation reaction to covalently bind to transpeptidases. However, unlike native stem peptide substrates where this bond is transferred to an acceptor peptide in the second step of the reaction, β-lactams bind to the enzyme and prevent it from synthesizing peptidoglycan (38). This acyl-enzyme complex can undergo slow hydrolysis (39) and regenerate the enzyme. However, in practical situations, the enzymes usually remain inhibited as β-lactams are intermittently administered during treatment. This results in inhibition of cell wall synthesis and eventual death of bacteria.

Historically, the sole targets of β-lactams were thought to be DDTs. Mtb possesses multiple proteins that have been identified as DDTs or their sequence homologs (29). However, recent studies have demonstrated that it is vital to inhibit both LDTs and DDTs (and potentially other proteins associated with peptidoglycan biosynthesis pathway) in order to completely inhibit peptidoglycan synthesis and kill Mtb (40, 41). While DDTs are known to be targeted by β-lactams, we are only beginning to understand how LDTs are inhibited. Crystal structures of a Mtb DDT bound by penicillin G (a penicillin), ceftriaxone (a cephalosporin) and imipenem (a carbapenem) provided direct evidence of binding, acylation, and inhibition of a DDT in this pathogen (42). Emerging evidence suggests that Mtb LDTs are not inhibited by penicillins (41) Some cephalosporins inhibit LDTs, but they are weaker inhibitors than carbapenems (43). Independent studies have reported thatcarbapenems bind more tightly and react most strongly with LDTs of Mtb compared to other β-lactam sub-classes(41, 44). Several laboratories have described crystal structures of LDTs of Mtb in complex with various carbapenems and penems, as well as their reaction kinetics (35, 39, 41, 45–50). Crystal structures are not currently available for LDTs bound to penicillins or cephalosporins.

In vitro activity of β-lactams

Penicillins in general are known to have poor activity against Mtb without protection by a companion β-lactamase inhibitor. In 1983, Cynamon and Palmer (51) described the activity of amoxicillin alone vs. amoxicillin plus clavulanate against 15 Mtb isolates in vitro. They found that amoxicillin alone exhibited no bactericidal activity at therapeutic doses, whereas the combination of amoxicillin/clavulanate was bactericidal against 14/15 isolates. Several subsequent studies similarly showed that the addition of a clavulanate or sulbactam (another β-lactamase inhibitor) to various penicillins, including ampicillin and ticarcillin, resulted in activity against Mtb that was not observed with penicillins alone (7, 8, 52, 53). Across all the reported studies, ampicillin or amoxicillin plus clavulanate resulted in the most potent activity of the combinations tested.

Cephalosporins exhibit in vitro activity against Mtb (54). The MICs of first-generation cephalosporins (such as cefadroxil and cefadrine) are within the therapeutic range, both alone and in combination with rifampicin and/or clavulanate against Mtb in vitro (55). A combination of ceftazidime (a third-generation cephalosporin) and avibactam (a β-lactamase inhibitor) has been reported to exhibit potency against growing and semi-dormant Mtb and also against DR-Mtb (56). Recently, Gold et al. described two novel cephalosporins that exhibit bactericidal activity against nonreplicating Mtb in vitro, are nontoxic, and stable in human plasma (57). Although cephalosporins have not been tested against Mtb in an animal model, their anti-tuberculous activities are compelling and warrant further study.

Carbapenems exhibit potency against Mtb even in the absence of a β-lactamase inhibitor. While their enhanced potency against Mtb was described in the 1990s (20), in 2009 Hugonnet et al. demonstrated that, unlike other β-lactams, carbapenems are slow substrates (and potentially slow inhibitors) of the β-lactamase BlaC, and exhibit potent activity against extensively drug-resistant strains of Mtb (5). Soon after, many laboratories began reporting potent in vitro activities of various carbapenems (8, 9, 44, 58, 59), including new experimental carbapenems (41). Recently, it was determined that carbapenems are unique among β-lactams, as they effectively inhibit Mtb LDTs (31, 35, 41, 44–48). In a simple experiment, investigators reacted Mtb LDTs with an equimolar mixture of various β-lactams and found that LDTs were exclusively acylated by penems and carbapenems and not by other sub-classes of β-lactams (41). Based on these results, this study proposed that carbapenems and penems are most effective against Mtb, not only because they inhibit DDTs and are less susceptible to inactivation by β-lactamase, but because they are the only β-lactam sub-classes that effectively inhibits LDTs. It should be noted that there are exceptions, as some cephalosporins are able to bind LDTs.

Compared to other β-lactams, some carbapenems appear less stable in water (60). This greatly influences the MIC, the most common measure of drug potency in vitro, especially in the case of Mtb, as MIC determination requires incubation with the drug for at least 10 days (61). Investigators have noted that MICs of carbapenems against Mtb and other mycobacteria, determined using standard broth dilution assay, can depend on how the drug is dosed (9, 20) and the timing of final assessment (62). Antimicrobial activities of various carbapenems and their mechanisms of action are reviewed in more detail elsewhere (60).

Penems are perhaps the least known amongst β-lactams, as they were developed only recently and there is currently only one drug from this class that is commercially available, faropenem. Penems differ structurally from carbapenems by the presence of a sulfur atom rather than a carbon atom in its bicyclic ring structure; resulting in improved chemical stability compared to older carbapenems such as imipenem (63). Faropenem was noted to have superior bactericidal activity over meropenem, including activity against a subpopulation of nongrowing, but metabolically active bacilli, which are thought to be responsible for relapsed infection following treatment (64). Additionally, a prodrug formulation, faropenem medoxomil, has improved oral bioavailability allowing for more convenient oral administration (65); however, this drug has not been FDA approved for use in the US.

Although significant insights have been gained in recent years with regards to various targets of β-lactams and the overall potency of each β-lactam sub-class against Mtb, we do not clearly understand the molecular mechanism subsequent to the inhibition of transpeptidases that triggers death of this organism when exposed to β-lactams. It is likely that additional enzymes are inhibited by β-lactams and each β-lactam inhibits each target at a different rate. While the specific details will have to await future studies, emerging evidence discussed above suggests the following broad generalization with regards to the anti-tubercular hierarchy, from most to least potent, of β-lactam sub-classes: carbapenems/penems > cephalosporins > penicillins.

Pharmacological properties of β-lactams

The mouse model has long been the de facto preclinical in vivo model for evaluation of new drugs and treatment regimens for TB. However, there is emerging evidence that this model underestimates the efficacy of β-lactams against Mtb as observed in human subjects (8, 64). The lack of efficacy in mice is related to inadequate drug exposure in serum, which is denoted by time above MIC (%TMIC). Renal dehydropeptidase-1 (DHP-1), an enzyme in the proximal tubules that is responsible for rapid clearance of many β-lactams, especially carbapenems, is much more active in mice than in humans (66). When evaluated in DHP-1-deficient mice, the efficacy of several classes of β-lactams increased significantly (66). This also helps to explain their apparent efficacy in humans, resulting in a three-fold higher %TMIC in serum (8). The addition of a DHP-1 inhibitor such as cilastatin, which is coformulated with imipenem, serves to further reduce renal drug clearance (67), although the adjunctive effect of cilastatin in TB treatment has not been clearly established.

Carbapenems and their potential for use in treating drug-resistant TB

Among carbapenems, the utility of meropenem for treating TB has been studied in some detail. Some of the major concerns cited with the use of meropenem are its chemical instability necessitating frequent dosing intervals, intravenous route of administration, and the need for its co-administration with clavulanate for optimal efficacy. Additionally, meropenem lacks efficacy in the chronic infection model, likely due to poor penetration and insufficient %TMIC in nonreplicating Mtb (8). This prompted the evaluation of newer carbapenems against TB, which have shown promising results. One study assessing in vitro activity of five β-lactams with or without a β-lactamase inhibitor against clinical TB isolates including MDR (multidrug-resistant) and XDR strains, showed that tebipenem, an orally bioavailable carbapenem, exhibited the most potent activity, with further reduction in MICs on the addition of clavulanate (58). Biapenem has been shown to inhibit growth of both drug-susceptible and DR-Mtb in vitro (59) and in vivo (68). Additionally, it is highly resistant to hydrolysis by DHP-1 as compared to meropenem (69, 70). A direct comparison between biapenem and faropenem using the mouse model of TB revealed biapenem to be superior (41). Emerging evidence of the anti-tuberculous activity of biapenem from in vitro and mouse studies, as well as its robust pharmacokinetics and safety data in humans (71), makes it a leading candidate among carbapenems to be considered for treatment of DR-TB. A clinical trial that directly compares meropenem and biapenem would be of high significance in identifying the carbapenem with highest utility for treating DR-TB.

It is vital to consider potential antagonism or synergy between existing anti-tuberculous antibiotics and β-lactams when developing new regimens for DR-TB. Not only are there no reports of antagonism between β-lactams and current TB drugs, two independent groups have reported synergy between various β-lactams and rifampicin, which is the cornerstone of treatment of drug-susceptible TB. Kaushik et al. described a high degree of synergy between carbapenems and rifampicin against both drug-susceptible and DR-Mtb in vitro (9). Based on this observation, synergy in vivo between these two antibacterials was anticipated. When evaluated in the mouse model of Mtb infection, a combination of biapenem and rifampicin was found to exhibit synergy against a drug-susceptible Mtb strain (68). However, the combination did not exhibit synergy against rifampin mono-resistant strains. It would be beneficial to assess if combinations of carbapenems and rifampicin maintain in vivo synergy against rifampicin sensitive, drug-resistant strains of Mtb. Ramon-Garcia et al. observed in vitro synergy between cephalosporins and rifampicin against Mtb (55). This combination has not been evaluated for synergy in an animal model. There are no published reports describing resistance to current first-line TB drugs conferring resistance to β-lactams. One study found drug-susceptible and XDR-Mtb strains to be equally susceptible to carbapenems and cephalosporins (5). In another study, a specific clade of XDR-Mtb strains from South Africa exhibited paradoxical hypersusceptibility to certain β-lactams (72).

It is clear from the literature that synergy is observed when combining a β-lactam with a β-lactamase inhibitor for treatment of DR-TB, yet few studies evaluating synergy between other antibiotic combinations have been performed. Based on our understanding of the differential transpeptidases present in mycobacteria, it stands to reason that a combination of two classes of β-lactams would exhibit synergy against Mtb. While penicillins and cephalosporins inhibit DDTs, carbapenems are more effective at targeting LDTs (41, 44). Therefore, a treatment regimen containing inhibitors of both DDTs and LDTs would potentially be more efficacious in disrupting peptidoglycan biosynthesis. For example, Gonzalo et al. found that the addition of amoxicillin to meropenem or meropenem plus clavulanate resulted in a reduction in meropenem MIC to within the clinically achievable drug exposures across 28 clinical MDR-TB strains in vitro (73). Indeed carbapenems also inhibit DDTs, but the empirical evidence demonstrating synergy between meropenem and amoxicillin is an indication that these two β-lactams inhibit different targets and together are more effective than when used alone.

Clinical trials of β-lactams against Mtb

Several anecdotal (25) and retrospective case series have been published over the past decade describing the efficacy of carbapenems in patients with MDR and XDR-TB. Compared to standard second-line treatments, patients receiving carbapenems had higher early treatment success rates and fewer adverse events; suggesting that carbapenems are viable options for treatment of DR-TB (21, 74–78). However, these studies tended to be small observational case series and long-term assessment of relapse-free cure was not performed. The results of these studies were recently summarized in a systematic review (26). The potential for deploying carbapenems for treatment of DR-TB has also been highlighted in a recent perspective (24).

An ex vivo assay called the whole blood bactericidal activity (WBA) was developed by Wallis et al to evaluate the efficacy of a drug in killing Mtb in humans (79). They demonstrated a correlation between WBA results and endpoints from traditional microbiological and clinical endpoints (80). In 2017, Gurumurthy et al. published data from a clinical trial analyzing WBA of antibiotic combinations against Mtb H37Rv (81). Healthy volunteers were treated with rifampicin alone and in combination with faropenem plus amoxicillin/clavulanate. Whole blood samples collected eight hours post-dose were inoculated with Mtb and analysis of colony-forming units was performed. They found that while faropenem plus amoxicillin/clavulanate had no bactericidal activity against Mtb, the combination of all three drugs was effective. The poor efficacy of faropenem without rifampicin was thought to be due to insufficient serum drug levels likely due to inadequate dosing of this drug. In contrast to this observation, a prior study had observed bactericidal activity of faropenem alone in an intracellular macrophage model of Mtb (64). These incongruent observations warrant additional studies to inform utility of faropenem for treatment of DR-TB in humans.

Recently, two phase-two randomized clinical trials have been initiated. In the first trial [NCT02349841], patients received either meropenem plus amoxicillin/clavulanate or daily treatment with standard isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) therapy. The EBA noted in both groups were similar, and adverse events were mild and infrequent among patients receiving β-lactam therapy (27). A second clinical trial comparing EBA of isoniazid, pyrazinamide, and faropenem plus amoxicillin/clavulanate is currently underway [NCT02381470]. The results of these studies will help to further our understanding of the practical role of β-lactams in TB treatment.

Conclusion

Recent studies show that we have not harnessed the full potential of β-lactams for treating DR-TB. For those β-lactams currently in clinical use, the pharmacokinetics and safety profiles have been well-established. Given the imminent need for additional treatment options against MDR/XDR-TB, it would be advantageous to move toward additional clinical trials as soon as possible. We can be optimistic in regard to β-lactam activity in humans against TB, as suggested by available preliminary clinical trial data. Providers are also currently limited in regards to therapeutic options, as they do not have access to several newer carbapenems that have demonstrated efficacy against MDR/XDR-TB. A push towards FDA approval for these agents will be beneficial. For patients whose treatment options are limited, β-lactams are readily accessible, safe to use, and affordable, and can be rapidly implemented in the clinical setting. This could mean the difference between life and death for patients suffering from drug-resistant TB, particularly in developing countries with limited financial and healthcare resources.

Acknowledgments

We thank Dr. P. C. Karakousis for critical reading of this manuscript. This work was supported by NIH grant R33AI111739. ESR was supported by the NIH T32 AI007291. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare no conflict of interest.

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