Deciphering the Role of β-Lactamase Inhibitors, Membrane Permeabilizers and Efflux Pump Inhibitors as Emerging Targets in Antibiotic Resistance

Abstract

Antimicrobial drugs have been noticed to have reduce activity effective due to upsurge witnessed in resistance of microbes. To deal with viewpoint of such a circumstance, we must seek ways to prevent it or atleast mitigate its effects in order to provide its activity against the microbes. Hence, novel antimicrobials are the one of the most promising solution for ending antimicrobial resistance. Furthermore, due to the less development of newer antimicrobials in recent years, the only way to combat microbial resistance are various synergistic approaches of exploring antimicrobial drug combinations. This combination's efficacy is due to a synergistic chemical that re-sensitizes the resistant microbial strain. It has been observed that classes of β-lactamases inhibitors, efflux pump inhibitors and membrane permeabilizers are of particular relevance, since they can break resistance to the most commonly used antimicrobials. This review explains the readers that how these synergistic combinations can help to reduce or eliminate the microbial resistance supported with clinical evidence.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-022-01045-6.

Introduction

Antimicrobials are agents act either by killing microorganisms or preventing their growth, depending on their site of actions. The common mechanisms are protein synthesis inhibition, cell wall synthesis inhibition, interference with DNA function, and interfering with intermediate metabolism [1, 2]. Penicillin, Sulphonamides, fluoroquinolones, and tetracyclines were developed and reported with antimicrobial resistance. The major source of hospital-acquired infections is Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecium, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter have shown high rate of resistance. According to literature, gram-negative infections may evolve multiple mechanisms of resistance[3, 4]. In 2014, the World Health Organization (WHO) noticed 4,80,000 new cases of multidrug-resistant tuberculosis (MDR-TB) and half of the patients were effectively treated[5].

Antimicrobial resistance refers to a drug becoming ineffective towards microorganism's which takes place due to genetic alteration. However, the indiscriminate use of antimicrobial drugs has widened the gap of antimicrobial resistance[4, 6, 7]. Microbes may adapt to multiple mechanisms to render antimicrobial agents ineffective. Degradation of antimicrobial agents by microbial enzymes, alteration of the target sites, decreased antimicrobial agent’s accumulation, and plasmid-mediated resistance are recognised as fundamental mechanisms of antimicrobial resistance[8]. Endogenous and Exogenous evolution can help the microorganism to develop resistance to antimicrobials. Former occurs when a microbial genome undergoes spontaneous mutation, resulting in resistance to a specific antibiotic [9, 10]. The transfer of a resistant gene from a resistant plasmid (R-plasmid) from resistant bacteria to susceptible bacteria termed as exogenous evolution. There are different steps for resistance mechanisms; Conjugation, transformation, and transduction. [11].

When two or more medications are used the result can be additive, sub-additive, antagonistic, or synergistic. The synergism mechanism not only improves the therapeutic capacity of the drug combination, but also reduces microbial resistance[12] and lowers the antimicrobial minimum inhibitory concentration (MIC) when used together. Synergism can be achieved through a variety of mechanisms; β-lactamase suppression, efflux pump inhibition and membrane permeabilizer which needs to be addressed [13–16].In this review, the focus is on synergistic agents and their effectiveness used to reduce antimicrobial resistance supported with clinical evidence (supplementary data).

β-Lactamase Inhibitor

β-lactamases are bacteria-produced enzymes which confer resistance to penicillin, cephalosporin’s, cephamycin, monobactams, and carbapenems. The gram-negative bacteria employ this as a primary defence mechanism to develop resistance[17, 18]. The β -lactamases are classified as the ambler and the Bush-Jacoby-Medeiros functional classification. The former is based on protein sequencing; spilt into A-D classes. All classes hydrolyse β-lactam rings, but A, C, and D use serine nucleophiles, whereas B requires a metal cofactor (zinc). Bush-Jacoby-Medeiros is based on substrate specificity; 2a prefers penicillin, 2b prefers penicillin and narrow-spectrum cephalosporins, 2be prefers penicillin, narrow spectrum and extended-spectrum cephalosporins and subgroup 2br enzymes are broad-spectrum β-lactamases with acquired resistance to clavulanic acid and associated inhibitors while retaining a subgroup 2b spectrum of activity. [19, 20].

The structure of β-lactam antibiotics β -lactam ring. The lactamase deactivates the molecule's antibacterial properties by breaking the β-lactam ring which can be prevented by a beta lactamase inhibitor. They could become substrates with a high affinity for the β-lactamase enzyme but unfavourable steric interactions. They can act as "suicide inhibitors," permanently inactivating the enzyme by secondary chemical reactions in the active region. There are a variety of β-lactamase inhibitors on the market, natural and synthetic. Individual β-lactamase inhibitors are not currently available on the market but β-lactam antibiotic with a similar serum half-life is combined with them[21, 22].

Clavulanic acid (Fig. 1a), β-lactamase inhibitor, was isolated from Streptomyces Clavuligerus [23], combined with amoxicillin and ticarcillin. This combination is effective against susceptible and resistant strains which are used to treat UTIs, pneumonia, ear, skin infections and soft tissue infections[24]. Sulbactam (Fig. 1b), as monotherapy is less effective, but it works better in ampicillin-sulbactam and cefoperazone-sulbactam combinations [11]. In vitro study of cefoperazone-sulbactam against different multidrug-resistant organisms (extended-spectrum producing β-lactamase (ESBL) producing E. coli and K. pneumoniae, carbapenem-resistant Enterobacteriaceae, P. aeruginosa and A. baumannii). The combination results decrease in MIC₅₀ value against multidrug-resistant organisms and susceptibility rate of treatment was higher as compared to cefoperazone alone. Furthermore, the susceptibility rate increases as the ratio of cefoperazone-sulbactam is changed from 2:1 to 1:1 and 1:2 for carbapenem-resistant Enterobacteriaceae and A. baumannii, ESBL producing k. pneumoniae [25].

Fig. 1.

Chemical strcuctures of BLIs. Structure a Clavulanic acid, b Sulbactam, c Tazobactum, d AAI101, e Avibactam

Tazobactam (Fig. 1c) is a penicillanic acid derivative; broader spectrum of activity as compared to clavulanic acid, used in combination with ceftolozane to treat infections. Ceftolozane is a new broad-spectrum cephalosporin with strong pseudomonal action. The combination of ceftolazane and tazobactam was approved for UTI and severe intra-abdominal infections[26]; effective against β-lactamases of classes A, C, and D and was evaluated against 1019 strain at MIC₅₀ and MIC₉₀ concentrations of 0.5 and 4 mg/L, respectively which was satisfactory. At an 8 mg/L dosage, 94.1 percent of strains' growth was inhibited[27].

Allecra Therapeutics' AAI101 (Fig. 1d) is a novel β-lactamase inhibitor; similar structure to tazobactam. It was demonstrated that the combination of AAI101-Cefepime was found to be active against various cefepime-resistant Enterobacteriaceae strains. The MIC₅₀ of cefepime was reduced from 64 mg/L for cefepime to 13 mg/L when given in combination with AAI101[28]. In another study, AAI101-Cefepime combination against 20 selected strains of K. pnemoniae and E. coli at various fixed concentrations (1:1, 1:2, 1:4, 1:8, and 1:16 mg/ml), MIC₅₀ value of cefepime was found to decrease with an increase in the concentration of AAI101 against different strains[29]. Avibactam (Fig. 1e) is a non-β-lactam based β-lactamase inhibitor that possesses good inhibitory activity against Pseudomonas aeruginosa. [30] having electrophilic carbonyl group. At 4 µg/ml, avibactam decreases ceftazidime's MIC₅₀ from 256 µg/ml to 0.25 µg/ml against K. Pneumonia and from 8 µg/ml to 4 µg/ml against P. aeruginosa. A study conducted on avibactam-ceftazidime combination against 20,709 Enterobacteriaceae strains gathered from US hospitals between 2011 and 2013; strains were found to be sensitive to avibactam-ceftazidime in 99.9% of cases. The MIC₅₀ and MIC₉₀ were 0.12 and 0.25 µg/ml, respectively[31].

Membrane Permiabilizers

Membrane permeation is important for the activity of the antimicrobial agent which is an issue with gram-negative bacteria having a distinct outer membrane that makes them naturally resistant to a wide range of antimicrobial treatments[32, 33]. The lipopolysaccharide layer (LPS) and underlying phospholipids prevent hydrophilic antibacterial drugs from passing through the outer membrane, whereas outer membrane proteins keep hydrophobic agents out. LPS is component of gram-negative bacteria's outer leaflet in the outer membrane; acts as a permeability barrier and is crucial in drug resistance. To improve antibiotic efficacy, researchers must find ways to promote drug diffusion and overcome the bacterial membrane barrier, which is responsible for antibiotic resistance. Permeabilizers are chemicals that weaken the outer membrane and increase the permeability of bacterial cells to exogenous products, such as antimicrobial drugs, in a nonspecific way. They disrupt the lipid part of the cell membrane as they dissolve the LPS layer, furthermore amplify the antibacterial activity of antibiotics that interact with intracellular targets and promote membrane permeability because of their lipophilic nature. In combination with antibiotics, the use of outer membrane permeabilizers may provide a further strategy for limiting gram-negative bacteria growth[34].

Polymyxins are a class of cyclic non-ribosomal polypeptides produced by the Bacillus genus (Fig. 2); made up of ten amino acid residues, with six of them being L-α, -diamino butyric acid (L-DAB) and DAB residues have numerous positively charged groups at physiological pH. They interact electrostatically with the outer membrane, displacing Mg²⁺ and Ca²⁺ cations from their binding sites, disrupting the integrity of the gram-negative bacteria's outer and inner membranes and causing leakage through the cell membrane. Azithromycin was found to be effective against the gram-negative rods due to the synergistic effect of colistin. When azithromycin was given in combination with colistin against A. baumannii, the bacterial cell entrance of the fluorescent dye SYTOX green increased by fourfold[35]. Study conducted on combination of colistin with meropenem/ sulbactam/ minocycline/ disodium Fosfomycin / and vancomycin has a synergistic effect against 23 carbapenem-resistant Acinetobacter baumannii strains. The strongest synergy was discovered between colistin and vancomycin (17.4% of strains), followed by colistin and minocycline (8.5% of strains), meropenem and fosfomycin (4.3% of strains) and no synergy between colistin and sulbactam[36].

Fig. 2.

General structure of polymyxin

Polymyxin B nonapeptide (PMBN), a cyclic peptide produced from Polymyxin B by proteolytic removal of its terminal amino acyl residue. It is less toxic and has no bactericidal action, but it still has the ability to permeabilize gram-negative bacteria's outer membranes, leaving them susceptible to antibiotics. At a dose of 5 mg/L, it improves the sensitivity of rifampin against E. coli, S. typhimurium, and P. aeruginosa by 30 times[37]. SPR741 is a cationic peptide produced from polymyxin B; carries three positive charges, resulting in a lack of fatty acyl tail. At 2 µg/ml concentration, it reduces the MIC of rifampin from 8 µg/ml to 4 µg/ml and clarithromycin from 64 µg/ml to 32 µg/ml[38]. Furthermore, it reduces the MIC of rifampin, retapamulin, fusidic acid, and clarithromycin by less than 32 folds[39].

Efflux Pump Inhibitor

Efflux pumps transport proteins are involved in the extrusion of hazardous substrates from within cells into the external environment. Gram-positive, gram-negative and eukaryotic organisms contain these proteins. In a difficult environment, this phenomenon ensures a cell's optimal development and survival[40]. Efflux transporters are categorized based on energy requirements as primary and secondary active transporters. To mediate drug efflux, primary active transporters are powered by the free energy obtained from the breakdown of adenosine triphosphate (ATP). Secondary active transporters, are related to drug extrusion to the influx of proton (H +) or sodium (Na +) ions across the cytoplasmic membrane via their electrochemical gradients. There are five major families divided by structure and substrate specificity viz; major facilitator superfamily transporters, resistance nodulation cell division transporters, ATP-binding cassette transporters, multidrug and toxic compound extrusion transporters and small multi-drug resistance transporters [41, 42].

Efflux pumps reduce intracellular drug concentrations to levels that are less harmful by constantly extruding antimicrobial medicines from a cell. In order to solve the antimicrobial resistance situation, clinically relevant bacteria with efflux-mediated drug resistance can over-express chromosomally-encoded efflux pumps[41]. Tet38 transporter (tetracycline) and CmlB (chloramphenicol); are antibiotic-specific. Several efflux pumps, lack substrate specificity and can expel a wide range of chemically or structurally unrelated molecules from the cell, resulting in MDR. Due to MDR the efficiency of front-line antimicrobial medicines in battling life-threatening illnesses has been reduced. The use of an efflux pump inhibitor will aid in the elimination of efflux pump-induced resistance[41].

Tetrandrine (Fig. 3a) is an efflux pump inhibitor isolated from Stephania tetrandrine; has synergistic effect with penicillin and fluconazole, boosting their concentration inside the microbial cell[11] and against C. parapsilosis (fluconazole/voriconazole). When compared to fluconazole alone (0.5 µg/ml), the MIC₈₀ decreased by 50% whereas Voriconazole's MIC₈₀ decreased, from 0.03 µg/ml to 0.02 µg/ml[43]. Synergistic effect of tetrandrine was observed with posaconazole against A. fumigatus isolates. Tetrandrine reduces the MIC of posaconazole from 0.0625–1 µg/ml to 0.0312–0.25 µg/ml, resulting in a 90% reduction in growth[44]. Reserpine (Fig. 3b) is a plant-derived efflux pump inhibitor isolated from Rauwolfia vomitoria roots. It improves the potency of tetracycline (a fourfold drop in the MIC) in two clinical isolates of MSRA (IS-58 and XU212) with Tet(K) efflux protein. It also inhibits the efflux of ciprofloxacin in E. coli and zone of inhibition caused by ciprofloxacin—reserpine in combination (19.0 ± 0.5 mm) is greater than that caused by ciprofloxacin alone (9.5 ± 0.5 mm)[45].

Fig. 3.

Chemical structures of EPIs. a Tetrandrine, b Reserpine, c Piperine, d 5'-MHC-D, e Epicatechin gallate, f Epigallocatechin gallate

Piperine (Fig. 3c) is an alkaloid isolated from Piper nigram that inhibits human P-glycoprotein ABC transporters via cytochrome P450-mediated mechanisms. Piperine and piperidine have been shown to inhibit the efflux pump in Mycobacteria and S. aureus. In S. aureus, piperine increases ciprofloxacin accumulation by suppressing the NorA efflux pump[46]. Piperine increases activity of rifampicin by altering uncharacterized efflux pump (Rv128c) in M. tuberculosis H37Rv. Aso, piperine lowers the MIC of ethidium bromide in Mycobacterium smegmatis, demonstrating that it can be used as an EPI across bacteria species[47]. A flavolignan, 5’-methoxy-hydnocarpin (Fig. 3d), is isolated from Berberis fremontii. It enhances the efficacy of NorA substrates (norfloxacin and berberine), by suppresing the efflux pump. When flavonolignan 5'-methoxyhydnocarpin-D (5’-MHC-D) given in combination with berberine, the antibacterial activity of berberine increases by 16 folds and MIC of berberine was reduced to 16 mg/L[48].

Epicatechin gallate (Fig. 3e) and epigallocatechin gallate (Fig. 3f) were isolated from the leaves and buds of Camellia sinensis. When combined with oxacillin, found to be effective against MRSA strains; EMRSA-16, EMRSA-15 and BB568. The MIC of oxacillin against EMRSA-15 is reduced by four to eight-fold by epigallocatechin and gallocatechin. Also, they reduced the MIC of oxacillin by less than 64-fold at a concentration of 25 mg/L against tested strains [49]. It was found that 20 µg/ml of epigallocatechin gallate decreases the MIC of norfloxacin by 4-folds against S. epidermis and S. aureus and found to have less inhibitory potential against the NorA transporter[50].

Conclusion

The increase in microbial resistance against antimicrobial agents is the major concern to health care professionals. As monotherapy is ineffective, the hunt for new approach is necessary to combat the microbial resistance. Synergistic antimicrobial combinations will help to decrease drug doses which minimize side effects and thus be a strategy to combat toxicity and resistance development issues.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This review did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declarations

Conflict of interest:

The authors declare no conflict of interest.

Ethical approval

Not applicable since it is a review article.