Antibiotic Adjuvants for Combatting Antimicrobial Resistance

Jan. 27, 2023

The threat of antimicrobial resistance (AMR) is, well, threatening. The gist of the story: bacterial adaptability and transmission of antimicrobial resistance genes, coupled with the overuse and misuse of antibiotics across societal sectors, has created a situation in which the antibiotics we have relied on to stave off infection are losing their power. Finding new antimicrobial compounds is one part of the solution to this growing problem—another is preserving the efficacy of antibiotics that already exist. 

Antibiotic Adjuvants: Compounds for Preserving Antibiotic Efficacy

Antibiotic adjuvants are compounds that show little-to-no antimicrobial activity by themselves. Rather, they synergize with antibiotics to minimize, or even block, bacterial resistance to restore and conserve the activity of antibiotics. 

There are a variety of mechanisms through which bacteria become resistant to antibiotics. In some cases, resistance is associated with an intrinsic cellular feature (e.g., the outer membrane, or OM, of gram-negative bacteria can make it difficult for antibiotics to enter the cell). Bacteria also develop resistance through adaptation, or via acquisition of genetic elements harboring AMR genes (e.g., plasmids). Regardless of how bacteria acquire the capacity to resist antibiotics, the methods they use to accomplish the task vary widely, including enzymatically inactivating drugs, shooting antibiotic molecules out of the cell via efflux pumps or modifying the drug so it is no longer effective. 

Mechanisms of antimicrobial resistance.
Bacteria can resist antibiotics in various ways. Some traits are intrinsic while others are acquired through mobile genetic elements, like plasmids.

Most antibiotic adjuvants that are under investigation or that are actively used in the clinic target bacterial molecules or cellular structures central to these mechanisms of resistance. To that end, there are 3 major classes of adjuvants that have been studied: β-lactamase inhibitors, efflux pump inhibitors (EPIs) and membrane permeabilizers, with the first showing the greatest clinical success to date.

β-lactamase Inhibitors: Quintessential Antibiotic Adjuvants

β-lactam antibiotics, including penicillins, cephalosporins and carbepenems, are some of the most widely used antibiotics, in part because of their broad antimicrobial scope (they can target both gram-positive and gram-negative bacteria). These drugs inhibit proteins involved in peptidoglycan biosynthesis (i.e., penicillin-binding proteins, or PBPs), leading to loss of cell viability and eventual cell lysis. However, many bacteria, including opportunistic pathogens like Klebsiella pneumoniae and Pseudomonas aeruginosa, bring their own weapons to the table in the form of β-lactamases.

Interactions between β -lactam antibiotics and β -lactam interactive proteins
Interactions between β -lactam antibiotics and β -lactam interactive proteins in gram-negative bacteria. β -lactamases intercept the antibiotic molecules so they can’t bind to PBPs.
Source: Bush K. and Bradford P.A./Clinical Microbiology Reviews, 2020.
These enzymes, often acquired via horizontal gene transfer, inactivate β-lactam antibiotics by severing the β-lactam ring inherent to their structure. While there are many different types of β-lactamases, they are split into 2 main groups: those whose activity depends on a serine residue in the active site (serine-β-lactamases) and those that use 1 or 2 zinc ions to catalyze the reaction (metallo-β-lactamases). 

Luckily, clinicians have several adjuvants (i.e., β-lactamase inhibitors) for dealing with β-lactamase-harboring bacteria. These compounds prevent bacterial inactivation of β-lactams in a couple of ways, including by binding and blocking the active site of the β-lactamase. They have been instrumental in maintaining the efficacy of β-lactams amidst spreading resistance. 

There are currently 6 β-lactamase inhibitors approved for clinical use: clavulanic acid, sulbactam, tazobactam, avibactam, varborbactam and relebactam, each with slight differences in chemical structure and activity. All compounds are administered in combination with a specific antibiotic. For example, clavulanic acid is always administered with amoxicillin while sulbactam is paired with ampicillin. The β-lactamase inhibitor-antibiotic pair used depends on the infection and the resistance profile of the organism causing the infection. 

Not all inhibitors are effective against every type of serine-β-lactamases, of which there are various classes, and none of the current inhibitors work against metallo-β-lactamases. Given metallo-β-lactamases confer resistance to all penicillins, cephalosporins and carbapenems, and their global incidence is increasing, scientists are diligently working to find compounds that effectively inhibit these evasive enzymes. Researchers are also identifying new inhibitory compounds that use mechanisms other than direct β-lactamase inhibition. For instance, a recent study published in mBio showed that the purine nucleosides guanine and xanothosine could re-sensitize methicillin-resistant Staphyloccocus aureus to β-lactam antibiotics by reducing cellular levels of the secondary messenger, c-di-AMP, which is required for β-lactam resistance.

Additional Antibiotic Adjuvants

Efforts to expand the pool of β-lactamase inhibitors are complemented by those aimed at developing other types of adjuvants, particularly EPIs and membrane permeabilizers. Adjuvants outside of these 2 groups have also been explored, including bacteriophages (i.e., viruses that infect bacteria) and compounds that target microbial response regulators (signaling proteins involved in numerous bacterial physiological processes).

Efflux Pump Inhibitors

Embedded in bacterial membranes, efflux pumps expel substances, including antibiotics, from inside the cell. Some efflux pumps burp out specific molecules, while others are non-specific and expel a diverse range of compounds. 

EPIs are adjuvants that restore antibiotic sensitivity by preventing resistant bacteria from shooting antibiotics out of cell, thus increasing their intracellular concentration and activity. This can occur by hindering assembly of pump components at the membrane, or by inhibiting expression of genes encoding pump components, among other mechanisms.

In the laboratory, the EPI, PAβN, is used to increase the potency of antibiotics toward gram-negative bacteria, though for various reasons, including toxicity concerns, it is not applicable for clinical use. Other compounds may show more promise. For instance, scientists showed that 4-hexylresorcinol (4-HR), a natural, plant-derived compound, reduced the minimum concentration of antibiotics needed to inhibit growth of bacteria like E. coli and S. aureus by 2-50-fold, depending on the antibiotic. Moreover, a combination of 4-HR and the antibiotic polymyxin B improved survival in a mouse model of K. pneumoniae-induced sepsis compared to the antibiotic alone.

Efflux pump
Bacteria can pump antibiotics out of the cell through efflux pumps.
Source: Schlenk/Wikimedia Commons

Still, unlike β-lactamase inhibitors, there are currently no EPIs used in the clinic. Advancement of EPIs through the clinical pipeline will depend, in part, on a better understanding of the function and substrates of EPIs in bacterial pathogens and how to target them. 

Membrane Permeabilizers 

To function, antibiotics generally need to pass through the bacterial membrane via diffusion or membrane channels (porins). This can be tricky, especially when faced with the additional OM in gram-negative bacteria. As such, compounds that promote diffusion of antibiotics across the membrane would be a welcome addition to the antibiotic adjuvant toolbox. Researchers recently found that a compound, known as NV716, binds lipopolysaccharides (LPS) on the OM of P. aeruginosa to permeabilize the membrane. In doing so, it re-sensitizes the bacterium to abandoned antibiotics (i.e., those that are not used against the microbe due to resistance, such as doxycycline, rifampicin and chloramphenicol). NV716 also acts as an EPI, highlighting that adjuvants may have more than 1 molecular mechanism.

Other potential permeabilizers come from everything from plants to sharks. Existing compounds may also be repurposed as membrane-disrupting adjuvants. For instance, the anti-malarial drug, mefloquine, was shown to alter membrane fluidity of S. aureus and potentiate activity of the β-lactam antibiotic, oxacillin, against methicillin-resistant and susceptible strains of the bacterium.

Antibiotic Adjuvants are a Single Piece of the AMR Puzzle

Antibiotic adjuvants are an important part of addressing the AMR crisis, but they, alone, are not enough. Rather, they are part of an interconnected web of tactics that include ensuring the strategical use of antibiotics specifically for bacterial infections and enhanced education about antibiotics for people across societal spheres, including those working in health care and agriculture. Searching for novel antimicrobials, along with bolstering our repertoire of adjuvants, will also be important for fostering a future in which treatable bacterial infections stay treatable.

To learn more about AMR, and how to combat it, check out our resource page.

Author: Madeline Barron, Ph.D.

Madeline Barron, Ph.D.
Madeline Barron, Ph.D. is the Science Communications Specialist at ASM. She obtained her Ph.D. from the University of Michigan in the Department of Microbiology and Immunology.