The growing menace of drug resistant pathogens and recent strategies to overcome drug resistance: A review

2.1 Limiting the uptake of drug

Drug uptake can be limited due to the difference in cell structures in different bacteria. Gram-negative bacteria are inherently resistant to antimicrobials due to the presence of an outer cell wall (Wanda, 2016). This mechanism is not seen in gram-positive bacteria due to the lack of cell walls. Hydrophobicity and hydrophilicity also have a part in limiting the uptake of drugs. Porins determine which compounds are allowed to enter the cell through the cell wall based on their hydrophilicity and hydrophobicity. Usually, large porins allow hydrophilic molecules to enter while hydrophobic molecules are stopped (Blair et al., 2014).

2.2 Modification of drug target sites

This is one of the most common mechanisms adopted by pathogens. By modifying the target site, the drug fails to interact with the target site properly or may not recognize the site at all. This is achieved through target protection and target modification (Munita and Arias, 2016). Target modifications can be achieved through point mutations in gene coding sites, enzyme-mediated alterations in the binding site, and replacement or bypass of the original target.

2.3 Inactivation of drug

It is achieved through complete drug molecule destruction or the addition of chemical groups to the drug. β-Lactamases are the most common example of drug destruction. They destroy the drug by hydrolyzing a site in the ring structure of β-lactam drugs (Wanda, 2016). In the case of the transfer of chemical groups, the methyl group is transferred to the drug. Acetyl, adenyl, and phosphoryl groups are some of the most commonly transferred chemical groups and acetylation is the most common reaction. It offers resistance against a wide range of antibacterials like aminoglycosides, chloramphenicol, and streptogramins (Wanda, 2016).

2.4 Efflux pumps

These are complex pieces of machinery capable of flushing out toxic compounds from the cell (Munita and Arias, 2016). They were first described in E.coli in the 1980 s (McMurry et al., 1980). Both gram-negative and gram-positive pathogens make use of this mechanism. Five types of efflux pumps have been described so far and they are major facilitator superfamily, small multidrug resistance family, Resistance Nodulation Cell Divison family(RND), ATP binding cassette family (ABC), and multidrug and toxic compound extrusion family (MATE). Another type of efflux pump has been added recently to the list making the total six. The sixth efflux pump is proteobacterial antimicrobial compound efflux (PACE). The ABC family derives its energy for transport by utilizing ATP. The other five utilize electrochemical energy trapped in transmembrane ion gradients and they are secondary active transporters(Du et al., 2018). The mechanism of antibiotic resistance by pathogens is shown in Fig. 1.

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Fig. 1

Mechanism of antibiotic resistance in pathogens.

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2.5 Mechanism of drug resistance in gram-negative and gram-positive bacteria

The differences in the mechanism of drug resistance in these bacteria primarily depend on their cell structure. The presence of a thick cell wall in gram-negative bacteria helps them in preventing entry of drugs through mechanisms like limited uptake of drugs (Wanda, 2016). On the other hand, gram-positive bacteria do not have this mechanism due to the lack of a cell wall. The thick cell wall in gram-negative bacteria makes it inherently resistant compared to gram-positive bacteria which lack cell walls (Breijyeh and Karaman, 2020). While gram-positive bacteria are naturally resistant to cell wall synthesis inhibiting β- lactamases. Enzymatic degradation of antibiotics by the production of β lactamases and decreasing the susceptibility and affinity to their target binding site, known as penicillin-binding protein (PBP), by the acquisition of exogenous DNA or by native changes in PBP are the two major mechanisms conferring resistance to gram-positive pathogens (Breijyeh et al., 2020) But in recent times, many specialized mechanisms have been described in different bacteria, which depends on their structure, surroundings, etc. In most cases, these mechanisms are variations of the efflux pumps due to mutations in their genes.

3.1 Narrow spectrum antibiotics

The use of narrow-spectrum antibiotics rather than broad-spectrum is another strategy used by clinicians these days to target AMR or MDR pathogens (Andrew, 2014). Combinatorial drug use has shown better results in the management of AMR. A pathogen might have developed resistance to the mode of action of a particular drug, but it is highly unlikely that it might have gained resistance to two different types of drugs with totally unrelated modes of action (Nosten and others, 2000). The need for combining antibiotics stemmed from the fact that no single antibiotic is effective against all infections universally. Many of the combinations at the beginning were opportunistic and had little data backing their efficacy or molecular mechanism. The advantages of antibiotic combinations were reported in the past. The combination of streptomycin with penicillin was reported in 1950 and trimethoprim with sulfonamides in 1968. These combinations had improved spectrum and efficacy compared to them being used individually. Combination drug therapy backed with clinical and epidemiological data are now a major clinical practice. Synergy and antagonism are the two factors on which drug combinations are built. They are calculated using the Fractional Inhibitory concentration Index (FICI) method in microbiological labs (Tyers and Wright,2019). These are some of the most common strategies adopted by clinicians all over the world to combat AMR under the guidance of WHO, CDC, and the rest.

Apart from these several national and international action plans have been put forward by several countries to control AMR. Some of the noted programs and agencies to combat AMR are National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS) in the USA, Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) in Canada, Observatoire National de l'Epidémiologie de la Résistance Bactérienne aux Antibiotiques (ONERBA) in France, and Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) in Denmark. Celebration of AMR week as ‘World Antibiotic Awareness Week’ in November is also being done globally every year to create more awareness among the public. This also helps and motivates health care workers and policymakers to adopt and engage in best practices to tackle and prevent further antibiotic resistance.

5.1 Antimicrobial peptides

Anti-microbial peptides (AMPs) or host defense particles are produced by all life forms from bacteria to higher organisms. The discovery of AMPs occurred during the extraction of an antimicrobial agent gramicidin from a Bacillus strain isolated from a soil sample. They have a broad spectrum of action, even against bacteria, fungi, and protozoa. Their amphipathic nature facilitates their insertion and interaction into cell walls. Even though their mode of action is through causing damage to the cellular membrane, they can also be employed to target different proteins like regulatory enzymes, DNA, and RNA. Since the chance of developing resistance to AMPs once they are introduced into clinical practice is high, more molecular level studies need to be done to understand their mode of action and resistance to these compounds to go ahead with AMPs as an alternative to antibiotics (Aslam et al., 2018).

5.2 Phage therapy

Phages are used against resistant bacterial pathogens. It is emerging as a very promising method against bacterial AMR. Phage therapy was introduced as a new therapeutic approach when bacteria like E. faecium, S. aureus, and K. pneumoniae started becoming resistant to the existing antibiotic compounds. The major advantages of phage therapy are i) inherent toxicity, ii) lack of cross-resistance with antibiotic classes, iii) they are strain-specific unlike the broad-spectrum antibiotics, and iv) they are effective against both gram-negative and gram-positive bacteria (Principi et al., 2019).

The demerits associated with phage therapy is the chance of treating them as foreign bodies by the immune system, they may also carry antibiotic resistance genes in them. Extensive studies are required to understand the genome of the phages and their properties for manipulating effectively depending upon our requirements.

5.3 Nanoparticles

Nanoparticles (NPs) falling under the 1–100 nm range are used for the treatment of antibiotic-resistant infections (Baptista, 2018). The advantage of the use of NPs in AMR treatment, it increases the drug stability and solubility inside the body. NPs are more target-specific and their small size makes their delivery into the system much easier. NPs also tackle the problem of poor membrane permeability of antibiotics. The use of NPs as an effective agent to tackle the problem of drug resistance cannot be immediately put into practice due to health concerns associated with it. NPs although a promising technique, have been reported to aid in the horizontal gene transfer of Antibiotic Resistance Genes (ARGs). Their excessive use can result in their introduction into the environment subsequently aiding in the transfer of ARGs from one bacteria to another (Su et al.,2019).

5.4 Plant secondary metabolites

Plants are rich in medicinal properties, making them ideal candidates for treating bacterial pathogens. Plants include a wide range of phytochemicals with antibacterial properties such as saponins, alkaloids, tannins, flavonoids, and others (Gupta and Birdi, 2017). Plant phytochemicals are a valuable source of bioactive compounds, have antimicrobial properties, and can reverse antibiotic resistance (Khameneh et al., 2019). Some of the plant secondary metabolites interfere with molecular targets like cell signaling(Gupta and Birdi, 2017).

5.5 Emulsions

It is used for the delivery of a particular drug or compound. Usually, they are administered as oil–water emulsions in the form of droplets. Essential oils have been known to have antibacterial properties and these are most commonly employed. (Franklyne et al. 2016). Among the 3000 known Essential Oils (EOs), 300are are commercially important. EOs have certain drawbacks, they are sensitive to oxygen, light, and moisture making them undesirable for use in pharmaceutical industries. These problems are addressed by nano-encapsulation. Nano-emulsions of EOs can be produced by this process which can increase the activity of these bioactive compounds and also increase their dispensability by increasing their cell permeability through the cell wall of pathogens(Hosna et al.,2019).

5.6 Biosurfactants and quorum sensing inhibitors

They are biologically active surface-acting molecules produced by microbes. Biosurfactants due to their structural specialties have a detergent-like effect on the cell membrane of bacterial pathogens. They can avoid bacterial colonization by acting as anti-adhesive agents and not letting bacteria stick to the surface thereby preventing colonization (Rivardo et al, 2009). Antimicrobial activity of biosurfactants from Bacillus subtillis multidrug-resistant (MDR) bacteria has already been reported (Fernandes et al, 2007).

Quorum sensing (QS) is a surveillance mechanism used by bacteria for colonization and infection. QS is done with help of certain signal molecules produced by the bacteria themselves, which accumulate and get recognized by a receptor on the bacterial surface (Miller and Bassler, 2001). QS is a mode of bacterial communication by breaking the QS by appropriate quorum sensing inhibitors, the process of bacterial colonization can be prevented. It depends on the density of the bacterial population. Once the signal molecules produced by bacteria passes the threshold level the respective receptor will get activated and bind to the signal molecule inducing various bacterial behavior such as biofilm formation, bioluminescence, virulence factor production, and others (Ni et al., 2021). Both gram-positive and gram-negative bacteria have different kinds of quorum sensing signals. In gram-negative bacteria, the Qs system is mediated by the signal molecule Acylhomoserine lactone (AI-1) and in gram-positive bacteria, it is Auto-inducing peptides (AIPs) and Furanosyl Borate diesters in both gram-positive and gram-negative bacteria. (Jiang et al, 2019). QS inhibitors do not kill the pathogen rather weaken their pathogenicity by inhibiting various processes like biofilm formation, production of virulence factors, and expression of pathogenic genes (Ni, et al., 2021). Quorum sensing inhibitors can be used with existing antibiotics as combinatorial therapy against resistant pathogens Breijeh and Karaman,2020).

5.7 Plasmid curing

Often the antimicrobial resistance genes (AGRs) are located on the plasmids. These are mobile and aids in the transfer of ARGs from one cell to another. This type of transmission is responsible for the global dissemination of MDR bacteria. They are typically found in gram-negative bacteria and code for genes such as ESBL, carbapenemases, and others. (Holmes et.al, 2016). Plasmids show a high degree of plasticity (Kado, 2014). Plasmid curing is a process wherein, a bacterium is made devoid of its plasmids which contain most of the resistance genes. This technique removes the resistance leaving the bacterial population intact and making the pathogens more susceptible to antibiotics or antimicrobials. This is advantageous to revive the gut microbiome as most of the antibiotics wipe them out along with the pathogens in the body that might have ended up in the body through the food chain. Plasmids curing agents can be administered to people who are traveling abroad to prevent the spread of AMR globally. It can also be given to patients before surgery to avoid contracting the risk of nosocomial infections. Since not many options are available at present, this technique can be exploited and marketed (Buckner et.al, 2018).

5.8 Reverse vaccinology

It utilizes the sequencing of whole genomes of pathogenic bacteria to discover genome encoded antigens which were impossible in the two centuries of vaccine history. Vaccines could be designed and developed with data from the computer instead of starting from culturing the microbes in labs. Four component Meningococcus B vaccine, which was recently licensed has proven that reverse vaccinology is an efficient way to select antigens that can confer resistance against antigenically variable bacterial pathogens. Antigens are selected based on their sequence conservation and predicted surface exposure. Then expressed as recombinant proteins and tested out in pre-clinical models for eliciting functional antibodies. The promising candidates are then combined in the final step to achieve a vaccine with the best possible spectrum coverage based on molecular epidemiology data. 4CMen B was recently introduced in the U.K in all infants and showed an effectiveness of 82.9% against all Meningococcus B strains (Parikh et.al, 2016).

5.9 Potentiation approaches

In this technique, the developing of efflux pump inhibitors and prioritizing potentiator mechanisms that affect targets on the outer surface of the bacteria rather than penetrating to the cytoplasm have been tried. The need for co-development of a potentiator along with the antibiotic has also been tried as an alternative approach. This method tries to circumvent the poor penetrability of antimicrobials due to the highly complex nature of bacterial cell walls and pumps incorporated in them. It is a rather attractive approach as an effective efflux inhibitor can broaden the spectrum of a gram-positive agent by making it susceptible to gram-negative bacteria(Baker et al. 2018). Potentiation of gram-positive selective antibiotics against gram-negative pathogens reported previously depended on the disruption of the outer membrane (Martin et al., 2019). Adjuvants are used to potentiate antibiotics. They are compounds with very little to no antibiotic activity but when combined with a drug it enhances the activity of the drug considerably. Adjuvants also bring down the demand for the immediate discovery of new drugs as they can revive existing antibiotics against resistant pathogens (Breijyeh and Karaman, 2020).

5.10 Active uptake approaches

In this technique, the antibiotic influx is increased through bacterial membrane transporters. Various bacterial membrane transporters like iron transporters and sugar are exploited to increase the influx into the cells. The antibiotic fosfomycin was tested by using the bacterial sugar transporters to enter the cells (Baker et al. 2018).

5.11 Dendrimers

Dendrimers, dendrons, and dendritic polymers are attractive due to their controlled synthesis and monodispersity. Dendrimers range in size from 1 to 10 nm and they can cross cell membrane due to their controllable size and with the help of lipophilicity of the skeleton. It can encapsulate drugs and deliver to target tissues not entangle at the higher molecular weight. These properties make them very useful to inhibit biofilm formation. In P. aeruginosa they prevent the Lec B- fucose interaction thereby preventing biofilm formation. They can also disperse formed structures and act as antimicrobial nano-carriers (Ortega et.al, 2020).

5.12 Targeting drug-resistant pathogens by actinomycete secondary metabolites

Actinobacteria have been a hot topic among researchers for a long time and this is mainly due to its potential to produce diverse active secondary metabolites. Actinobacterial secondary metabolites have been known to be useful against not only pathogens but also proved to be useful in the agriculture sector and many other industries. Approximately 20,000 biologically active secondary metabolites have been discovered so far of which approximately 13,700 are reported as biologically active compounds and nearly 10,000 are antimicrobials which include antibacterial, antiviral, and antitumor compounds (Berdy, 2012). Of the actinobacterial species, the species Streptomyces alone contributes nearly 7600 compounds with the potential to suppress multi-drug resistant pathogens.

Anti- Quorum sensing or inhibition is the newest and most promising aspect of actinobacteria. It prevents biofilm formation in pathogens (Miao et.al, 2017). These are compounds that can inhibit the expression of virulence factors through the production of enzymes capable of breaking down signaling molecules and quorum sensing peptides (Betancur et.al, 2017). Diverse compounds derived from actinomycetes are effective against MDR pathogens.

Antimicrobial peptides are another promising secondary metabolite from the actinomycetes. They are oligopeptides with a varying number of amino acids. It was first described in 1939. Bacteriocin is a potent peptide isolated from Actinomycetes. They have many immunological activities including antimicrobial activity (Richard et.al, 2015). There have been several reports of antimicrobial peptides from actinomycetes in recent years. Antimicrobial peptides derived from actinomycetes are given in Table1.

5.13 Host-directed therapies (HDTs)

It enhances protective immune signatures, reduces exacerbated inflammation, or balances immune reactivity at the infection site. HDTs also exploit the repurposing of drugs commonly used in therapy. Under HTDs selective pressure on the microbe is avoided and, hence, the risk of developing resistance to treatment is also less thereby it paves way for new therapy (Puccetti et al., 2020).

Some other approaches which include supplementation of micronutrients, immune-modulators, and antimicrobial peptide inducers are suggested to be beneficial. Prolonged intake of antibiotics interferes with the human microbiome thereby weakening the immune system and the host becomes susceptible to pathogens. Hence, restoring microbiota by probiotics, prebiotics, and synbiotics approaches has become an attractive option for potential therapeutics (Puccetti et al., 2020).

5.14 Postbiotics

Deals with the role of microbiota-derived metabolites, high-throughput sequencing, and metabolomics studies lead to the identification of a series of postbiotic compounds that can contribute to directly and specifically manipulate the microbiota and host functions. It includes lipids, carbohydrates, proteins, organic acids, vitamins/co-factors, peptidoglycan-derived muropeptides, or lipoteichoic acids, or immunomodulators, anti-inflammatory, hypocholesterolemic, anti-obesogenic, anti-hypertensive, anti-proliferative, and antioxidants (Roger and Dragsted, 2019). The pros and cons of alternative approaches for the treatment of MDR pathogens are given in Table 2.