Antimicrobial resistance a continued global threat to public health – A perspective and mitigation strategies

Alteration of drug targets

Antibiotic resistance in bacteria can be attributed to various structural or compositional changes in the target sites of bacterial cells, as observed in the study by Lade et al.^[28] In addition, the presence of separate target protection molecules can prevent antibiotics from effectively binding to their targets, providing a protective shield against the action of antibiotics.^[29] For instance, GPB, particularly polymyxin-resistant strains, can develop drug resistance through alterations in antibiotic-binding targets, such as in S. aureus, which acquires resistance by converting its penicillin-binding protein (PBP) to PBP2a, which is encoded by the mecA gene. The recent discovery of mecB, mecC, and mecD genes has further compounded the understanding of antibiotic resistance.^[30] PBP2a exhibits low-affinity binding to β-lactam antibiotics and other classes of antibiotics, including macrolides, lincosamides, aminoglycosides, glycopeptides, oxazolidinones, and lipopeptides.^[27] The emergence of antibiotic resistance has posed challenges to the use of vancomycin and clindamycin in complicated urinary tract infections and skin-soft-tissue infections caused by MRSA. The emergence of vancomycin-resistant S. aureus and clindamycin-resistant MRSA has significantly reduced the therapeutic efficacy of these antibiotics.^[28,31] Enterococci utilize van genes to modify the D-Ala-D-Ala dipeptide at the C-terminus of the translocated pentapeptide to significantly reduce the binding affinity of vancomycin, rendering it less effective against Enterococci.^[32] In addition, methylation of the 16S or 23S ribosomal RNA (rRNA) can lead to the loss of efficacy of antibiotics targeting ribosomes, as it disrupts binding affinity by causing steric clashes.^[33] To achieve the drug resistance target, protection proteins can act through three mechanisms: direct contact to dislodge the antibiotic, allosteric conformational changes in the target to dislodge the antibiotic, or enabling the target to function despite the presence of the bound antibiotic.^[34] Antibiotic resistance ATP-binding cassette proteins (F-subtype) can rescue stalled ribosomes by inducing conformational changes in 23S rRNA, leading to antibiotic dissociation.^[35] These diverse mechanisms [Box 2] of antibiotic resistance underscore the complexity of the problem and emphasize the need for innovative strategies to combat the challenge effectively.

Enzyme-mediated drug inactivation

Bacteria have produced various enzymes as part of their defense mechanisms against antimicrobial drugs, which render these drugs ineffective by inactivating their action. Three examples of such enzymes are β-lactamase, aminoglycoside inactivating enzyme, and aminoglycoside modifying enzyme. These enzymes fall into different categories, namely, hydrolases, passivation enzymes, and modified enzymes, respectively.^[30] The mechanisms of resistance may be further exemplified by highlighting the below-mentioned enzymes.

Alterations in outer membrane permeability

β-lactam antibiotics generally penetrate the outer membrane of GNB through hydrophilic channel proteins, primarily porins, which facilitate the diffusion of small molecules including nutrients and antibiotics. In P. aeruginosa, various resistance mechanisms have been identified, among which intrinsic resistance, particularly the absence or modification of the outer membrane porin OprD, is extensively studied. OprD is a substrate-specific porin that mediates the uptake of carbapenems, such as imipenem and, to a lesser degree, meropenem.^[47] However, loss or inactivation of OprD leads to reduced susceptibility to imipenem and, to a lesser extent, meropenem in P. aeruginosa.^[47] This loss of function can result from mutations, deletions, or nucleotide substitutions in the oprD gene. Chromosomal mutations that inactivate OprD reduce carbapenem binding to their target proteins, such as PBPs. The impact of OprD inactivation is more pronounced for imipenem than for other carbapenems such as doripenem, meropenem, and ertapenem. Therefore, mutations that affect porin channels or reduce their expression contribute to reduced susceptibility to β-lactams in bacteria, with low outer membrane permeability playing a crucial role in its intrinsic resistance.^[48] Furthermore, alterations in the constricting loops of porin channels can influence the diffusion of polar drugs and mutations in these regions selectively obstruct antibiotic passage.^[49] The co-existence of carbapenemases and OprD loss complicates diagnostics and limits treatment options. MspA, the major porin of Mycobacterium smegmatis, plays a crucial role in the uptake of hydrophilic antibiotics. Studies have shown that deleting the mspA gene significantly increases resistance to various agents,^[50] indicating the importance of porin channels in uptake of drugs or inducement of resistance [Box 5].

Active efflux mechanism

The efflux pumps or transmembrane protein complexes are found in clinically significant bacteria. Active efflux is alternatively recognized as the efflux pump system or drug pumping system. The insufficient drug concentration allows bacteria to withstand the impact of the medication, causing drug resistance.^[30] Efflux transporters can facilitate the movement of diverse antibiotics out of bacterial cells and the most significant efflux transporters are major facilitator superfamily, ATP-binding cassette, multi-drug and toxic compound extrusion, small multi-drug resistance, and resistance-nodulation-division (RND).^[27] RND efflux system falls in the intrinsic resistance mechanism and is the most significantly prevalent superfamily. The primary RND efflux pumps include MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY/OprM (OprA). This efflux pump includes proteins such as AcrB in E. coli, MexB in P. aeruginosa, and MtrD in N. gonorrhoeae, as well as CmeB and CusA, which provide resistance to heavy metals.^[51] The three major components of these pumps are as follows: (i) Efflux Transporter is an inner membrane protein of the bacterial cell responsible for capturing the substances (e.g., antibiotics) from inside the cell or from the inner membrane itself. (ii) Outer Membrane Channel is the pathway that expels captured substances from the cell, moving them from the inside to the outside environment. (iii) Accessory Protein (Periplasmic) connects the efflux transporter and the outer membrane channel that ensures proper communication and coordination between the two, allowing the substances to be efficiently transported out of the cell. Efflux pumps, such as the MmpL and MmpS systems, contribute to intrinsic resistance in M. tuberculosis by actively expelling drugs. Active efflux pump is also involved in gram positive bacteria in inducible antibiotic resistance. Their proficiency in expelling antibiotics has turned them into an effective mechanism for AMR [Box 6].^[52]

Targeting AMR enzymes

Several effective approaches have been explored to target AMR enzymes. These approaches include the CRISPR-Cas system, RNA silencing, essential oils, Small Molecules-Improved Chemical Entities, phytochemicals, and enzyme inhibitors.^[56,58,59] The CRISPR-Cas system, renowned for gene editing, can target AMR genes in bacteria and disrupt these genes, rendering the bacteria susceptible to antibiotics again. It is a bacterial defense system which utilizes RNA-guided, DNA-encoded, or DNA-targeting mechanisms to defend against the intrusion of bacteria by mobile genetic elements and foreign genetic material, including plasmids and phages. Another promising approach involves RNA silencing, where binding of complementary cis and trans sequences to the regulatory regions of mRNA prevents the synthesis of proteins during translation. Antimicrobial compounds obtained from plants sources as well as the essential oils have been found useful as alternative strategies to control bacterial infections. Essential oils are known to exhibit potent antimicrobial activity, while Small Molecules-Improved Chemical Entities are the novel direct-acting small molecules which may emerge through the improvement of existing antibiotics or by the production of new compounds with innovative targets and mechanisms of action.^[55,59] Phytochemicals or secondary metabolites of plants have shown potential as antibacterials both on their own and in combination with other antibacterial agents which are considered as a significant alternative treatment. Enzyme inhibitors are the entity intended to specifically inhibit activity of AMR enzymes, thereby restoring the efficacy of antibiotics. Figure 3 represents various approaches for targeting AMR enzymes.

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Figure 3:

Various approaches for targeting antimicrobial resistance enzymes.

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Antibiotic combination therapy

Antibiotic combination therapy and amalgamating antibiotics in novel regimens embraces to be a valuable approach in combating drug-resistant infections. The concept of antibiotic synergy refers to the amplified effectiveness of antibiotics when combined at the optimal ratio.^[60] When certain drugs are judiciously chosen and combined in the accurate proportion, the bactericidal activity becomes synergistic having substantial ability in killing bacteria resistant to antibiotics. Such a therapy has previously been practiced in the treatment of TB, infection by human immunodeficiency virus and also in other non-infective aliments like chemotherapy of cancer. The synergistic amalgamation therapy can be established reasonably for application in patients harmlessly as the component of such medicines is well identified.^[60] One well-established example is the combination of a β-lactam antibiotic with an aminoglycoside, which has been extensively utilized to treat various Gram-negative bacterial infections. Another effective approach involves combining antibiotics with β-lactamase inhibitors.^[61] β-lactamase inhibitors prevent the breakdown of β-lactam antibiotics by the bacterial enzymes, thus enhancing their efficacy and expanding their spectrum of activity. In recent times, the combination of antibiotics with biocides has also emerged as a successful therapeutic strategy.^[62] In TB, meropenem, when combined with clavulanic acid, shows potential for treating susceptible TB as it is effective against non-replicating forms of bacilli, which are challenging to eliminate even with standard first-line drugs such as INH and RIF. Furthermore, a constant and repetitive synergy has been observed by combining various antibiotics with plant extracts, which has increased the inhibitory effects against bacteria compared to using antibiotics alone.^[63] Combination therapy is an effective approach, as shown in Figure 4, and can lead to enhance treatment outcomes against resistant bacteria.

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Figure 4:

Combating antimicrobial resistance through antibiotic combination therapy. AMR: Antimicrobial resistance, RNA: Ribonucleic acid, GNB: Gram-negative bacteria, TB: Tuberculosis, CRISPR: Clustered regularly interspaced short palindromic repeats

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Drug delivery system

Researchers are actively exploring various approaches to combat AMR bacteria. One promising avenue is the use of drug delivery systems, such as synthetic siderophores and polymeric nanoparticles, which have shown potential in enhancing the effectiveness of antibiotics.^[64] Innovative techniques, such as antibody-drug conjugates and CRISPRCas systems, target AMR genes in plasmid with the aim of reducing resistance in the bacteria.^[65] Another alternative approach involves antivirulence compounds, like quorum sensing inhibitors, which can disrupt bacterial communication and coordination, thus reducing bacterial pathogenicity.^[66]

Unconventional approaches

Researchers have explored various alternative drugs against AMR infections, including antihistaminic, non-steroidal anti-inflammatory drugs, anesthetics, antipsychotics, and cardiovascular drugs. One promising approach is fecal microbiota transplantation (FMT), which has shown high efficacy in treating recurrent Clostridium difficile infections by restoring a healthy gut microbiota.^[67] FMT has proven to be effective in displacing VRE particularly when it is the dominant microorganism within the gut microbiota.

Physicochemical methods

Physicochemical methods for combating AMR offer innovative approaches to address drug-resistant microorganisms.^[59] One such method is Atmospheric Pressure Non-Thermal Plasma, which effectively targets bacteria, viruses, fungi, and parasites with minimal side effects compared to conventional treatments.^[68] Photoinactivation or photodynamic antimicrobial chemotherapy (PACT) is another promising technique that utilizes visible light and photosensitizers to generate reactive oxygen species (ROS), effectively killing bacteria. PACT shows potential in combating AMR with localized application and reduced risk of resistance development.^[69] Metal oxide nanoparticles, including Silver, Iron (II, III) oxide, Titanium dioxide, Copper oxide, and Zinc oxide, demonstrate potent antibacterial effects through ROS generation. However, their use requires caution due to potential environmental impact and co-selection risks.^[70] Sonodynamic antimicrobial chemotherapy is another physicochemical method that utilizes ultrasound and sonosensitizers to efficiently and precisely kill microorganisms while minimizing side effects.^[71] Physicochemical strategies are considered as an advanced and effective mitigating approach [Figure 5].

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Figure 5:

Advanced physicochemical strategies as mitigating approach for antimicrobial resistance (AMR). APNTP: Atmospheric Pressure Non-Thermal Plasma, ROS: Reactive Oxygen Species, PACT: Photoinactivation or photodynamics Antimicrobial Chemotherapy, Ag: Silver, Fe₃O₄: Iron (II,III) oxide, TiO₂: Titanium dioxide, CuO: Copper oxide, ZnO: Zinc oxide, SACT: Sonodynamic Antimicrobial Chemotherapy.

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Vaccines

Vaccines directly reduce resistant pathogens and limit AMR spread. Innovative approaches such as reverse vaccinology and combining vaccines with antibiotics can target resistant strains.^[72] Vaccines affect resistance pathogens directly by lowering the incidence of infection and indirectly by lowering the spread of AMR-resistant strains among non-resistant species.^[56] Vaccination complements antibiotics in curbing AMR.

Systematic approaches

In September 2016, Ministry of Health and Family Welfare of India has established the National Action Plan on AMR (NAP-AMR) which is a comprehensive strategy to combat AMR, through collaborative efforts of three technical bodies, namely, Intersectoral Coordination Committee, Technical Advisory Group and Core Working Group. The WHO’s Global Action Plan on AMR, which was approved by the World Health Assembly in 2015, serves as a major foundation for the NAP-AMR.^[73] Antimicrobial Stewardship (AMS) is crucial for ensuring antibiotics that are used only when necessary. The WHO advocates AMS programs to promote responsible antibiotic use, emphasizing the correct dose, reason, and duration of treatment. The introduction of the Antimicrobial Stewardship Program (ASP) was well-received for its systematic approach. However, the widespread use of broad-spectrum antibiotics during the COVID-19 pandemic has exacerbated antibacterial resistance, particularly among MRSA and ESBL-producing bacteria. This has transformed healthcare facilities into hubs for MDR organisms. ASP aims to enhance the recognition, implementation, and innovation in antibiotic use, with future priorities in focus. Despite progress, such as expanding ASP to substantial number of secondary hospitals and establishing the Indian Council of Medical Research-AMR Surveillance Network, India faces significant challenges due to limited resources and laboratory capacity.^[74] Key achievements include the formation of a network of 20 tertiary care hospitals for AMR surveillance, including 14 government and 6 private institutions^[75], which include various initiatives [Box 7], required for harmonious work.

Combating AMR is a collective endeavor that demands a multifaceted strategy, involving the discovery of new drugs, responsible antibiotic use, improved diagnostics, robust infection prevention, continuous monitoring, and international cooperation to effectively address and prevent the spread of resistance.