Penicillin was the first β-lactam antibiotic discovered and the second antibiotic to be used in human medicine after the sulfonamides. The group contains several natural and semisynthetic compounds and their pharmacokinetics and spectra of action differs significantly. Penicillin G (benzylpenicillin) and Penicillin V (phenoxymethylpenicillin) are the two natural penicillins used in human medicine mostly used to treat Gram-positive bacterial infections (Kulkarni et al., 2019). Among the semisynthetic penicillins, aminopenicillins like ampicillin, developed by the addition of an amino group to the benzylpenicillin, are the most clinically used antibiotics. Amoxicillin, developed by adding hydroxyl group to ampicillin was among the first broad spectrum penicillins that displayed activity against both Gram-positive and Gram-negative bacteria due to its better water solubility and absorbed from gastrointestinal tract. However, emergence of penicillinase producing bacteria prompted researchers to look for novel molecules that resisted penicillinase activity and thus methicillin was developed. Sooner, isoxazolyl penicillins which included the oxacillin, cloxacillin and dicloxacillin also known as antistaphylococcal penicillins, which had better oral absorption than methicillin were introduced to treat infections caused by Penicillin G resistant staphylococci (Smith et al., 1962). In short order, several other semisynthetic penicillins such as carboxypenicillins, acylureidopenicillins and amidino penicillins with improved action and bioavailability were developed, collectively known as the extended spectrum penicillins.

The cephalosporin antibiotics have a dihydrothiazine ring fused to the β-lactam nucleus with a 6-membered sulfur containing ring. Cephalosporins are the most frequently prescribed class of β-lactam antibiotics because of their broad spectrum of activity and lower allergenic and toxicity risks (Bush and Bradford, 2016). The use of cephalosporins extensively improved the treatment of infectious diseases and drastically reduced morbidity and mortality rates in pneumonia, meningitis, secondary infections in cancer patients, complicated skin and soft tissue infections, urogenital infections, infective endocarditis, serious bone and joint infections and Salmonella infections in children (Kwak et al., 2017; Kapil et al., 2019). Based on the spectra of action, cephalosporin groups of antibiotics have been classified into five generations.

Aztreonam is the most used monobactam antibiotic (Groman, 2009). Monobactam antibiotics are characterized by a single β-lactam ring not fused to another ring like other β-lactams. These antibiotics were identified to be more effective against the Gram-negative bacteria as compared to the Gram-positive bacteria due to their lower affinity to PBPs (Brogden and Heel, 1986). Surprisingly, monobactam antibiotics are resistant to bacterial metallo-β-lactamases (MBL), but susceptible to serine β-lactamases (SBL). Interestingly, Blais et al. had recently reported the discovery of a novel monobactam antibiotic, LYS228 effective against both MBL and SBL producing Enterobacteriaceae (Blais et al., 2018). The antibiotic is now in phase II clinical trials in patients with UTI and intra-abdominal infections.

Penems, the broad spectrum β-lactam antibiotics are majorly synthetic in nature and are sub-grouped as alkylpenems, aminopenems, arylpenems, oxypenems and thiopenems which are distinguished by the side chain of the unsaturated five-membered ring. Carbapenems are the most common penem antibiotic used which differs from other groups by possessing double bond between C2 and C3 and a substitution of carbon for sulfur at C1 (Papp-Wallace et al., 2011). One among the most used carbapenem antibiotics is the amidine derivative of thienamycin, Imipenem (N-formimidoyl-thienamycin) which is a broad spectrum β-lactam antibiotic effective against both Gram-positive and Gram-negative bacteria. However, imipenem is rapidly degraded by kidney dehydropeptidase-I (DHP-I). Hence, another broad spectrum carbapenem antibiotic, meropenem was developed which was relatively stable to DHP-I and that possessed enhanced activity against Enterobacteriaceae, P. aeruginosa, Haemophilus influenzae, and Neisseria gonorrhoeae. Other newly developed carbapenem antibiotics include doripenem, panipenem and biapenem (Perry and Ibbotson, 2002; Goa and Noble, 2003; Greer, 2008).

β-Lactamases are a diverse group of enzymes that have the ability to hydrolyze the amide bond of the β-lactam ring rendering the antibiotic ineffective. β-Lactamase producing E. coli was first identified in 1940’s even before the first β-lactam antibiotic was introduced for clinical use. Currently, there are numerous β-lactamases identified that have narrow or broad-spectrum activity against different β-lactam antibiotics. The sequence information of 7,537 β-lactamases, 1,526 structural information, and kinetics of 47 β-lactamases evolved over the years have been listed in the Beta-Lactamase DataBase (BLDB) revealing the diverse nature of the enzymes (Naas et al., 2017). These enzymes are usually located in the periplasmic space of the bacteria and they hydrolyse the β-lactam antibiotic before they reach the target molecule. Many Gram-negative bacteria also produce membrane vesicles that bear high levels of these enzymes that act on the antibiotics and reduce the concentration even before it reaches the infection foci (Ciofu et al., 2000).

Various characteristics like molecular structure, substrate profile, biochemical properties and functional characteristics were initially used to classify the β-lactamase enzymes. The Amber classification classifies the enzymes into four different classes (class A, B, C, and D) based on its sequence motifs and hydrolytic properties. Class A, C, and D constitute enzymes that employ their serine residue for the nucleophilic attack and hydrolyze of the β-lactam antibiotic while class B enzymes utilize a metal-activated water nucleophile to drive the hydrolytic reaction. There have been various reviews that summarize the different classes of β-lactamases and their substrates (Page, 2002; Nagshetty et al., 2021; Akhtar et al., 2022). Some β-lactamases have been identified to be specific to certain species of bacteria and some have been identified to be successful in global dissemination and confer selective fitness to bacterial clones (Fernández et al., 2012). Many ESKAPE pathogens have been identified to possess more than one class of β-lactamase resistance gene rendering them multidrug resistant and hard to treat (Oliveira et al., 2020). In 1980s, Enterobacteriaceae isolates capable of hydrolysing extended spectrum β-lactam antibiotics were reported from around the world and were identified to possess extended spectrum β-lactamases (ESBLs) such as TEM, SHV, and CTX-M. Carbapenem antibiotics were the treatment options for such ESBL producing isolates. However, bacterial isolates resistant to carbapenem antibiotics soon evolved capable of producing carbapenemase enzymes. Genes that confer ESBL activity (bla_TEM, bla_SHV, bla_CTX-M) and carbapenamase activity (bla_KPC) in the class A, carbapenamases (bla_NDM, bla_VIM and bla_IMP) in the class B, ESBLs (bla_CMY, bla_AMP and bla_ADC) in class C and the carbapenemase encoding bla_OXA genes in class D have been explicitly been successful in transmission among the Enterobacteriaceae family, A. baumannii and P. aeruginosa (Raible et al., 2017; Ahmed et al., 2020).

These classes of resistance genes are either chromosomally encoded or plasmid encoded. Plasmid encoded resistance genes have demonstrated significant success in the global spread of AMR among bacteria in clinical and non-clinical settings. KPC encoded by bla_KPC gene has been identified to have the potential for inter-species and geographical dissemination due to its association with Tn-3 type transposon Tn4401 capable of inserting into different types of plasmids of Gram-negative bacteria (Arnold et al., 2011). Additionally, the global success of KPC dissemination is also attributed to the clonal expansion of K. pneumoniae ST258, ST512, ST304, and ST11 (Munoz-Price et al., 2013). Mostly, bla_NDM variants are plasmid encoded and are rapidly transferred between enterobacterial isolates K. pneumoniae and E. coli. NDM encoding isolates are resistant to almost all β-lactam antibiotics with tigecycline and colistin being the only treatment options (Wu et al., 2019). Recently, there have been various reports of chromosomal integration of usually plasmid encoded β-lactamase resistance genes like bla_CTX-M and bla_OXA-48 in E. coli isolates (Rodríguez et al., 2014; Turton et al., 2016). In many other cases, co-existence of more than one class of β-lactamase resistance gene in both chromosome and plasmid was observed in Gram-negative bacterial pathogens (Poirel et al., 2010). The β-lactamase resistance genes have been found associated with various mobile genetic elements (MGEs) like plasmids, transposons, insertion sequences (IS) and integrons. bla_IMP-1 has been identified to be associated with Class I integrons which may have chromosomal or plasmid location in different species (Watanabe et al., 1991; Arakawa et al., 1995). Verona integron-encoded metallo-β-lactamase (VIM) is another prevalent integron associated MBL followed by German imipenemase (GIM-1) and Seoul imipenemase (SIM-1) located in class 1 integrons of Pseudomonas spp. Insertion sequences like ISAba1, ISAba4 and transposons such as Tn2006 and Tn2008 are found associated with the bla_OXA-23 gene in A. baumannii chromosome while ISAba3 and IS18 are found associated with bla_OXA-58 (Poirel and Nordmann, 2006; Corvec et al., 2007). Recently Chen et al., reported the identification of a novel ESBL encoded by the gene bla_OXA-830 in the chromosome of environmental bacteria Aeromonas simiae (Chen et al., 2019). The OXA-48 like carbapenemases in Enterobacterales has been found to be associated with various transposable elements and insertion sequences. Klebsiella pneumoniae and E. coli circulating in the North Africa and Middle East have been reported to possess OXA-48 associated with Tn1999 variants on IncL plasmids (Giani et al., 2012). OXA-181 and OXA-232 endemic to the Indian subcontinent and few sub-Saharan African countries are associated with ISEcp1, Tn2013 on ColE2, and IncX3 types of plasmids. Additionally, clonal dissemination of certain high risk clones of K. pneumoniae (ST147, ST307, ST15, and ST14) and E. coli (ST38 and ST410) are also a minor cause of the spread of OXA-48 like carbapenemases (Pitout et al., 2019). Many ESBL genes such as bla_TEM, bla_SHV, bla_CTX-M, carbapenemase genes such as bla_SPM, bla_IMP, bla_VIM, bla_NDM, bla_KPC and bla_OXA-48 are conjugative plasmid encoded in ESKAPE pathogens which allows in the easy transmission of these genes among the bacterial isolates (Kim et al., 2011; Veeraraghavan et al., 2019). Thus, β-lactamase enzymes form the most common method of β-lactam resistance in clinical pathogens.

The PBPs are the target of the β-lactam antibiotics. The PBPs are transpeptidases or carboxypeptidases involved in peptidoglycan metabolism. Hence PBP modification serves as another major mechanism by which bacteria resist the action of the antibiotic. The mutational alterations in PBPs can confer resistance to β-lactam antibiotics (Chesnel et al., 2002). Unemo et al., in 2012 had reported the isolation of a high level cefixime and ceftriaxone resistant Neisseria gonorrhoeae having a penA mosaic allele with an additional A501P mutation in the PBP2 (Unemo et al., 2012). Moya et al. had previously reported an unusual mechanism of β-lactam resistance by mutational inactivation of non-essential PBPs (PBP4) coded by dacB which behaves as a trap target for β-lactams (Moya et al., 2009). Additionally, the mutation on dacB also triggers over-expression of AmpC coding β-lactamase enzymes and activating the CreBC (BlrAB) two-component regulator which together results in high level β-lactam resistance. Mutation in the PBP coding gene down regulates its expression which in turn reduces the peptidoglycan turnover. The reduced peptidoglycan synthesis is considered as a cue for over production of β-lactamase enzymes. Recently, Lazzaro et al. reported high-level resistance of Enterococcus faecalis to Ceftobiprole (BPR), a new-generation cephalosporin as a result of alterations in the pbp4 gene (Lazzaro et al., 2022). The mutations revealed to over-express PBP4 leading to increased transpeptidation causing higher peptidoglycan synthesis that prevented Ceftobiprole penetration into the bacterial cell. Thus, mutations leading to gain of function or loss of function of PBP have been determined to play a major role in β-lactam resistance in bacteria.

Gram-negative bacteria possess the OM that acts as an additional barrier for the transport of solutes into the cell. The selective transport of molecules is achieved by specialized proteins called porins present on the OM. Modulation of porin expression is another mechanism of β-lactam resistance particularly in Gram-negative bacteria. Various insertional elements and point mutations in the promoter and protein coding sequences have been identified to alter porin protein expression and structure. Escherichia coli mutants lacking OmpC and OmpF proteins were documented to demonstrate high level resistance to cefoxitin and other cephalosporin antibiotics (Jaffe et al., 1982). Similarly, characterization of meropenem resistant Serratia marcescens isolates revealed insertional inactivation of ompF gene by an IS element, IS1 (Suh et al., 2010). Mutations in ompK35 and ompK36 in K. pneumoniae and ompC and ompF in Enterobacter spp. have been associated with increased resistance to the majority of carbapenem antibiotics (Doumith et al., 2009). Pagel et al., noticed that mutations at the L3 loop of OmpU in the cholera causing pathogen, Vibrio cholerae resulted in decreased susceptibility of the bacteria to cephalosporin antibiotics (Pagel et al., 2007). Likewise, several studies utilizing either site directed mutagenesis, knockdown or knockout of porin encoding genes in several clinically important pathogens revealed that these pathogens could gain reduced susceptibility to even last generation cephalosporins and carbapenems (Smani et al., 2014; Choi and Lee, 2019). A recent study by Khalifa et al. revealed that 93.3% of E. coli and 95.7% of K. pneumoniae isolated from hospitals in Egypt were identified to have lost their porins or showed modified porins resulting in heightened resistance to all carbapenem antibiotics (Khalifa et al., 2021). Interestingly, there have also been reports of association of porin regulation and expression of efflux pumps. Carbapenem resistance in P. aeruginosa has been primarily due to a combination of decreased transcriptional expression of OprD and upregulation of MexAB-OprM active efflux system (Muderris et al., 2018). Thus, in clinical isolates, interrelation of different mechanisms of resistance has been identified and mutations in some genes have been identified to activate multiple other resistance pathways.

Decreasing passive influx and increasing active efflux of antibiotics is a common mechanism of antibiotic resistance in bacteria. In clinically important pathogens, especially Gram-negative bacteria, a large number of promiscuous/polyselective efflux pumps such as the Resistance-Nodulation-Division (RND) efflux pumps that can recognize and expel a large variety of antibiotics have been identified. Unlike the β-lactamase coding genes that are mostly acquired, genes coding for efflux pumps are mostly intrinsic and found in both susceptible as well as resistant clones. However, acquisition of mutations at the regulatory region, promoter or open reading frame (ORF) of the gene leads to over-expression of these genes that cause resistance. Though the role of efflux pumps in β-lactam drug resistance was widely described in P. aeruginosa, it was not until recently that its role was identified in the members of the Enterobacteriaceae family (Glen and Lamont, 2021). About 12 different RND efflux pumps have been identified in P. aeruginosa of which MexAB-OprM, MexXY-OprM, and MexCD-OprJ play important role in β-lactam resistance and at least one of these efflux pumps have revealed increased expression during antibiotic treatment (Masuda et al., 2000). RND efflux pumps have also been reported in K. pneumoniae conferring β-lactam drug resistance. KexD expression was identified to be high in imipenem resistant K. pneumoniae and KpnEF (SMR type efflux pump) over-expression was noticed in cephalosporin resistant isolates (Srinivasan and Rajamohan, 2013). Uddin and Ahn recently reported multiple efflux pumps (AcrAB, AcrEF, EmrAB, MdfA, and MdtK) involved in conferring reduced susceptibility of β-lactam antibiotics in Salmonella Typhimurium (Uddin and Ahn, 2018). The expression of efflux pumps has been identified to be highly increased in bacterial pathogens during antibiotic treatment thereby significantly increasing the MIC of the drug required for the treatment. The treatment of Klebsiella aerogenes with imipenem was identified to drastically increase the expression of the marA gene that codes for an efflux pump protein (Bornet et al., 2003). Many other efflux pumps that cause co-resistance to other antibiotics have been identified to co-spread with β-lactam resistance genes (Li et al., 2019). The OqxAB, a multi-drug efflux pump widely attributed to resistance against quinolones, nitrofurantoin and chloramphenicol is co-transferable with bla_CTX-M, rmtB and aac(6′)-1b etc. The oqxAB genes are located either on the chromosome and/or on plasmids and are flanked by IS26-like element in clinical isolates of Enterobacteriaceae which makes them transferable (Li et al., 2019). Efflux pumps that expel out multiple antibiotics thus posing threat of multidrug resistance (MDR) in bacteria have been a major concern to both researchers and clinicians alike.

The BLIs are compounds that render the β-lactamase enzymes produced by the bacterial isolates inactive to hydrolyze the antibiotic. The emergence of β-lactamase producing isolates were reported from clinical settings soon after the introduction of β-lactam antibiotics. Though several macromolecular fractions (MM4550, MM13902, MM17880, BRL1437) from different Streptomyces sp. were identified to pose β-lactamase inhibitory activity, they were not clinically translated due to their unstable nature and undesirable pharmacological profiles. Reading and Cole in the late 1970s reported the detection of a compound with novel clavum structure isolated from Streptomyces clavuligerus ATCC 27064 (Reading and Cole, 1977). The compound when used in combination with β-lactam antibiotics significantly enhanced its antibacterial activity against penicillinase and cephalosporinase producing E. coli, K. aerogenes, Proteus mirabilis, and S. aureus isolates. This compound identified to be clavulanic acid was the first clinically potent BLI. Soon, a combination of amoxicillin and clavulanic acid (Amoxiclauv/Augmentin) was used clinically and found highly successful against class A β-lactamase producing bacterial isolates. The similarity of stereochemistry between clavulanic acid and β-lactamase enzymes was identified as the major contributing factor for its potentiation activity (Reading and Cole, 1977). Some BLIs are by themselves β-lactam antibiotics that at sub-inhibitory concentration augment the activity of the partner β-lactam antibiotic.

Following the identification of clavulanic acid, the β-lactamase inhibitory activities of various synthetic penicillin-based sulfones were screened which led to the discovery of sulbactam and tazobactam (YTR 830; Akova, 2008). Sulbactam, the halogenated derivatives of penicillanic acid (6-β-bromo-and 6-β-iodopenicillanic acid), was identified to be effective against class A β-lactamase producing Gram-negative isolates (Rolinson, 1991). However, it was identified to be less potent as compared to clavulanic acid in its activity against S. aureus β-lactamases and bacteria that possess TEM variants. While sulbactam was combined with ampicillin, amoxicillin, piperacillin, cefoperazone, cefotaxime and cefepime, tazobactam is used in combination with piperacillin and ceftolozane. The piperacillin-tazobactam combination was identified to be effective against broad-spectrum β-lactamases-producing and some extended-spectrum β-lactamases-producing Enterobacteriaceae. However, the combination was not effective against AmpC β-lactamases producing Gram-negative bacteria (Rolinson, 1991; Gin et al., 2007). Ceftolozane-tazobactam combination was identified to be even effective against Pseudomonas. As a therapeutic value addition, recent preliminary in vitro studies by Sakoulas et al., revealed that tazobactam potentiate peptide antibiotic, daptomycin against MRSA and colistin resistant A. baumanii (Sakoulas et al., 2017). An interesting finding by Urban et al. revealed that different concentrations of the BLIs have different targets. Clavulanic acid, sulbactam and tazobactam at lower concentration were identified to manifest the potentiation activity by inhibiting the β-lactamase activity, while at higher concentration, it exerted its potentiation activity through PBP modulation (Urban et al., 1995). Important BLIs, their class, inhibition spectrum and clinical status are listed in Table 1.

Recently, two new BLIs of the diazabicyclooctanones (DBOs) group namely avibactam and relebactam were identified with enhanced carbapenamase inhibitory activity. They were the first non β-lactam BLI to be used clinically and they exerted their β-lactamase inhibitory activity by acylating the β-lactamases reveribily unlike clavulanic acid, sulbactam or tazobactam that exerted irreversible inactivation of the resistance enzyme (Ehmann et al., 2012). Avibactam potentiated ceftaroline, ceftazidime, aztreonam while relebactam was used in combination with imipenem (Yahav et al., 2020). Interestingly, relebactam at the same concentration potentiates imipenem by its β-lactamase inhibitory activity and also PBP inhibitory activity (Blizzard et al., 2014). Successive to DBO BLIs, several boronic acid based BLIs were developed which were found to be effective against β-lactamase producing bacteria and they are at different stages of clinical trials. Most of the boronic acid based BLIs were identified to have dual mode of action like the relebactam. They exert their potentiation activity by β-lactamase inhibition and also PBP modification thereby enhancing their spectrum. Boronic acid based BLIs were active against most of the Class A, C and D (Oxa-48) β-lactamases (Lepak et al., 2019; Liu et al., 2020). Some boronic acid based BLIs such as zidebactam (WCK 5222) and taniborbactam (VNRX-5133) were active against Gram-negative organisms producing various carbapenemases, including MBLs producers (Lepak et al., 2019; Liu et al., 2020).

Though there are various BLIs developed against SBL (Amber classes A, C and D), effective MBL inhibitors are sparse. Despite a large amount of research being carried out to identify a potent MBL inhibitor, very few have reached the clinical trials and none have been commercialized. Elores (CSE-1034: ceftriaxone+sulbactam+disodium edetate) is the only MBL inhibitor used in patients with ESBL/MBL producing bacterial infections. The drug is approved in India by the Indian FDA after phase III clinical trials (Chaudhary et al., 2017). The major mechanism by which MBL inhibitors exert their activity is metal chelation. Elores showed high treatment success (83%) against both ESBL as well as MBL producing pathogens and lower rate of adverse effects (8.4%) caused by other drugs of choice to treat the infections (Chaudhary et al., 2017). However, long term usage of sodium edetate is not advisable as the chelating agent could interfere with eukaryotic enzymes that utilizes divalent cations for their activity. Aspergillomarasmine A, a natural compound discovered to have inhibitory activity against NDM-1 and VIM-2 exert its action by chelating the zinc ion present at the enzyme’s active site (King et al., 2014). Bisthiazolidines and small bicyclic compounds also utilize the same mechanism and were found to be effective against B1, B2, and B3 MBLs (Hinchliffe et al., 2016). Currently, development of covalent inhibitors that can bind to specific conserved amino acids at the active site of MBLs are gaining momentum. Irreversible inhibition of MBL (IMP-1) by 3-(3-mercaptopropionylsulfanyl) propionic acid pentafluorophenyl ester by binding to Lys224 and activity of ebselen against NDM-1 by binding to Cys221 are examples for such strategies (Kurosaki et al., 2005; Chiou et al., 2015). The major MBL inhibitors have been recently reviewed by Li et al. (2022). Ceftazidime-avibactam, ceftolozane-tazobactam, ceftazidime-tazobactam, meropenem-vaborbactam and imipenem-relebactam are the recently developed β-lactam-BLI combinations used to treat ESBL and carbapenemase producing bacterial pathogens (Yahav et al., 2020). However, very limited BLIs have been identified that pose both SBL and MBL inhibitory activity and the quest for such compounds continues.

Efflux pumps are a mechanism by which bacteria counteract the action of antibiotics by pumping out the drug thereby significantly decreasing its concentration and effectiveness. EPI or blockers have grown as a potential and attractive strategy to overcome bacterial resistance on account of the dwindling novel antibiotics. Efflux pump blocker, Phenylalanyl arginyl β-naphthylamide (PAβN) has been widely explored for its potentiation activity in clinical pathogens possessing polyspecific efflux pumps (Lamers et al., 2013). 1-(1-napthylmethyl)-piperazine (NMP), another putative EPI was identified to potentiate oxacillin antibiotic by inhibiting direct binding of the substrate to the efflux pump (AcrAB and AcrEF) of pathogens in the Enterobacteriaceae family. However, the compound did not have action on E. coli isolates and MexAB-OprM or MexCD-OprJ efflux pumps of P. aeruginosa (Bohnert and Kern, 2005). Further, artesunate, an anti-malarial drug was identified to possess EPI activity by binding to AcrAB-TolC of E. coli and thereby potentiating several β-lactam antibiotics such as penicillin G, ampicillin, cefazolin, cefuroxime and cefoperazone (Li et al., 2011). A plant based 5,6,7-trihydroflavone EPI, Baicalein, was identified to potentiate β-lactam antibiotics including oxacillin, cefmetazole and ampicillin while Conessine, another plant-based compound interfered with MexAB-OprM efflux pump in MDR P. aeruginosa potentiating cefotaxime antibiotic (Siriyong et al., 2017). Karumathil et al., had identified the potentiation efficacy of trans-cinnamaldehyde, isolated from cinnamon, eugenol isolated from clove, carvacrol isolated from oregano oil and thymol obtained from thyme against MDR A. baumanii. The natural compounds exerted their action by acting on the AdeABC efflux pump of A.baumanii making it sensitive to seven β-lactam antibiotics toward which it was previously resistant (Karumathil et al., 2018). Catechin gallate, a component from tea extract, was identified to potentiate oxacillin against MRSA isolates by reducing their MIC from 64–512 μg mL⁻¹ to ≤0.5–1 μg mL⁻¹ (Stapleton et al., 2004). However, no EPIs which specifically potentiate β-lactam antibiotics have been commercialized and clinically used. Today, dual potentiation therapy by combining EPI-antibiotic combinations with antimicrobial photodynamic inactivation (APDI) that utilizes a photosensitizer to activate reactive oxygen species within the bacteria in presence of visible light and kill the pathogen are gaining popularity to target MDR and XDR strains (Tegos et al., 2008; Hamblin, 2016).

Permeabilizers are compounds that debilitate the OM thereby allowing the antibiotic to enter the bacterial cell and exert its action. Unlike the BLIs, research on β-lactam potentiators that act by permeabilizing OM and cell membranes of bacterial pathogens are at its infancy. There have been sparse studies that have identified OM permeabilizers that work in conjunction with β-lactam antibiotics though it is a promising strategy. Several natural compounds have been screened for its OM permeabilizing ability against β-lactamase producing and carbapenamase producing bacterial pathogens. Recently, Farrag et al. demonstrated the permeabilizing ability of gallic acid against β-lactamase producing P. aeruginosa (Farrag et al., 2019). The irreversible OM destabilization ability of gallic acid was determined to be the result of chelation of divalent cations from the OM and lipopolysaccharide components of the pathogen. A combinatorial potentiating strategy by combining an OM permeabilizing agent, a BLI and a suitable β-lactam antibiotic has been hypothesized to be an effective way to overcome infections caused by β-lactam resistant pathogens (Farrag et al., 2015). Interestingly, many of the BLIs that have been identified and clinically used also have PBP binding and modifying activity (Moya et al., 2017). Zidebactam and WCK 5153 have been identified to specifically bind to PBP2 apart from inhibiting class A, C and some class D β-lactamases which together enhance the activity of β-lactam antibiotic (Moya et al., 2017). Anti-parasite compound pentamidine was identified to potentiate Gram-positive drugs against the Gram-negative pathogen A. baumanii in animal models through a mechanism of membrane destabilization (Ando et al., 2012). Polymyxin antibiotics and nanopeptide derivatives of polymyxin B were identified to portray significant OM permeabilizing activity thereby potentiating β-lactam antibiotics (Vaara, 2019). Apart from the BLIs that exert OM permeabilizing activity, nanopeptide named NAB74 developed from polymyxin B has entered the initial phases of clinical trials making the strategy encouraging and optimistic.

Apart from the classical β-lactam potentiators, many natural, synthetic, and semisynthetic compounds have been identified to potentiate β-lactam antibiotics against β-lactamase producing bacteria. Few promising β-lactam potentiators discovered within the last 5 years include farnesol, bulgecins, thiosemicarbazones and octyl gallate. Synthetic structural variants of farnesol, a sesquiterpene alcohol was identified to potentiate ampicillin and oxacillin against MRSA strains both in-vitro and in-vivo (Kim et al., 2018). Farnesol have been identified to upregulate and downregulate genes involved in cell membrane biogenesis and efflux pump proteins of Gram-negative pathogens A. baumannii and E. coli (Kostoulias et al., 2016). However, the exact molecular targets and mechanism by which these compounds potentiate β-lactams against MRSA are not elucidated. Bulgecins which are iminosaccharide secondary metabolites of Paraburkholderia acidophila have been reported to be a potential β-lactam enhancer against β-lactam resistant P. aeruginosa strains. The putative mechanism of ceftazidime and meropenem potentiation by bulgecin is understood to be the inhibition of periplasmic enzymes, Slt, MltD and MltG lytic transglycosylases that are very important for bacterial cell wall synthesis (Tomoshige et al., 2018). Additionally, diaryl and dipyridyl-substituted thiosemicarbazones were identified to possess synergistic effects with carbapenem antibiotic, meropenem against broad spectrum of MBL possessing bacterial pathogens (Zhao et al., 2021). Dipyridyl-substituted thiosemicarbazones competitively and reversibly inhibited NDM-1. The inhibition of MBLs by thiosemicarbazones was identified to be by the Zn(II) binding ability of a sulfur group in the compound (Li et al., 2021). Octyl gallate, a FDA approved antioxidant was identified to potentiate β-lactam antibiotics, penicillin, ampicillin and cephalothin against MRSA by increasing the bacterial cell wall permeability (Tamang et al., 2022). The galloyl moiety of octyl gallate is identified to interact with multiple targets in the bacterial cell wall which inhibit effective cell wall synthesis. Though the in-depth mechanistic evaluation of these molecules is still under evaluation, the differential targets of these compounds other than the conventional β-lactam drug targets make them potential futuristic β-lactam potentiators.

Colorimetric assays that analyze the growth inhibition of the pathogens in presence of antibiotic, compound and combinations of antibiotic and compounds is a conventional method of potentiator screening. Microwell plates are used to screen multiple compounds at a time. Reporter strains that over-express the enzyme or protein against which the compounds are being screened and chromogenic substrates that allow easy detection of the desired enzymatic activity are used to develop the assays. Compounds are initially screened for their potentiation activity and further assays are performed to characterize their mode of action. BLIs, EPIs and OM permeabilizers can be identified by this technique. Colorimetric assay with CENTA and Nitrocefin as chromogenic substrate revealed the antibacterial activity of Syzygium aromaticum oil and fresh juice of Ocimum sanctum leaves against ESBL producing E. coli (Shrivastav et al., 2019). A high throughput whole cell turbidity assay was used for screening a 645,000 compounds library against AmpC overproducing reporter strain to identify small molecule inhibitors of AmpG which is necessary for the induction of AmpC (Li et al., 2016). The study discovered 8 potential AmpG inhibitors that potentiate the activity of cefoxitin antibiotic inhibiting the growth of ESBL producing P. aeruginosa (Collia et al., 2018). Modified whole cell colorimetric assays such as the UV–Vis spectroscopy that can evaluate real time activity of β-lactamases and inhibitory activity of β-lactam potentiators are used widely for screening potential antibiotic adjuvants (Yang et al., 2017).

Molecular docking and molecular dynamic simulations (MDS) are very popular and well-established techniques for virtual screening of compounds. In-silico predictions not only predict the mechanism of resistance and identify novel potentiators, but also help in understanding structural restructuring capabilities for enhancing compound activity. During the screening of lead molecules, parameters of GRID Molecular Interaction Fields (MIFs) such as H-bond donor, H-bond acceptor and hydrophobicity are inspected to identify favorable active sites in the protein, and the molecule which gives the highest interactions is selected. Several studies have used in-silico screening to identify BLIs and EPIs that potentiate β-lactam antibiotics. FLAPdock (Fingerprint for Ligands and Proteins) is one such molecular docking tool that has identified a number of potential molecules active against class A β-lactamases (CTX-M, KPC-2, AmpC), class C β-lactamases, and MBLs (NDM-1 and VIM-2; Spyrakis et al., 2020). Ten natural compounds exhibiting potent MBL inhibiting activity targeting NDM-1 were identified by in-silico screening using Autodock Vina and Schrödinger suite software Extra Precision (XP; Spyrakis et al., 2020; Salari-Jazi et al., 2021). In another recent study, in-silico screening using Auto Dock4 software revealed Carsonic acid to exhibit promising inhibitory effects against NDM-1 (Yang et al., 2020). In-silico predictions thus are a promising strategy to identify broad-spectrum BLIs and EPIs though their in-vitro success has to be analyzed through various other assays.

By being able to analyze massive amounts of data instantly, machine learning (ML), artificial intelligence (AI), and neural networks (NN) have ushered in a golden era of drug discovery and synthesis. Bhadra et al., had used ML to analyze distribution of amino acid sequences in AMPs to predict their novel antibacterial and potentiating activities (Bhadra et al., 2018; Cunha et al., 2021). Mansbach et al. used ML coupled with the Hunting Fox Algorithm to study the membrane penetration activity of compounds in MDR P. aeruginosa (Mansbach et al., 2020). ML integrated with high-throughput Fourier-transform infrared spectroscopy has been applied to detect novel β-lactam resistance genes and β-lactam potentiators (Cunha et al., 2021). Parvaiz et al. specifically screened compounds potentiating β-lactam antibiotics by targeting bla_CMY genes from a 700,000 compounds library by using Site-Identification by Ligand Competitive Saturation (SILCS) technology, Ligand-grid Free Energy (LGFE) analysis and ML based random-forest (RF) scoring (Parvaiz et al., 2021). Thus, the intersection of ML and AMR has enabled the development and enhancement of several models that have aided new antibiotic and antibiotic potentiator discovery.

In many cases, antibiotic potentiating molecules which are not synthetic are either derived from microorganisms itself such as secondary metabolites, plant based such as polyphenols or alkaloids or animal based such as AMPs. The presence of such antibiotic potentiating molecules could be detected by various omics techniques. The major omics techniques utilized for novel drug and potentiating molecule discovery include genomics, metagenomics, transcriptomics, proteomics and metabolomics.