Lantibiotics as Alternative Therapeutics

Lantibiotics as Alternative Therapeutics explores alternative therapeutics, lantibiotics and other novel drugs. This book provides concrete information to readers regarding lantibiotics and various types of antimicrobial peptides with their mode of actions in treating various multidrug resistant organisms. It explains various techniques that are involved in analyzing antimicrobial peptides and their mode of actions. The development of antibiotic resistance has now reached a point of crisis where innovative methods and application of novel compounds and methods are required to prevent the spread of drug resistant infections.

Novel compounds exhibit different modes of action to the currently used mechanism of therapeutics in order to combat against the resistant organisms. Lantibiotics hold considerable potential as a consequence of their unusual structure, unique mechanisms of action and their potency against multi-drug resistant bacteria. This book will be useful for pharmaceutical industry scientists and researchers in microbial and biomedical research as well as graduate and advanced students in microbiology, medical biotechnology, health, and pharmaceutical sciences.

• Includes the biology, molecular interaction with target molecule, putative genes and analytical techniques to isolate and identify compounds
• Incorporates relevant case studies to increase understanding
• Focuses on recent trends on novel antimicrobial agents and antibiotic resistance research
• Discusses new arena of diseases, apart from acute and chronic infections

• Language: English
• Publisher: Academic Press
• Release date: Feb 17, 2023
• ISBN: 9780323991421

Book preview

Preface

Sanket Joshi, Rajiv K. Kar, Dibyajit Lahiri and Moupriya Nag

The development of antibiotic resistance has now reached a point of crisis, and innovative methods and application of novel compounds and methods are required to prevent the spread of drug-resistant infections in both cases of community and nosocomial environments. Such novel compounds exhibit different modes of action compared to the currently used mechanism of therapeutics to combat resistant organisms. The production of ribosomally synthesized antimicrobial peptides produced by bacteria is also referred to as bacteriocins that act as an alternative to the existing antibiotics. The various types of peptides consist of diverse groups comprising Class I bacteriocins, which are mostly the modified amino acids, after translation, which have developed great importance as pharmaceutical products. The groups of lantibiotics exhibit their activity predominantly against the Gram-positive group of bacterial cells, which also includes various drug-resistant organisms such as methicillin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus, and vancomycin-resistant Enterococci. Different groups of the antimicrobial peptides demonstrated excellent in vivo activities and have resulted in clinical evaluation for the treatment of life-threatening diseases. Although they possess various positive attributes, they also possess some limitations that include instability, insolubility at physiological pH, low production levels, and susceptibility to proteolytic degradation. The implementation of various technologies comprising genome mining, as well as high-throughput screening strategies for both in vivo and in vitro expressions, has provided a large amount of information relating to the existence of various types of structurally and functionally diverse lantibiotics.

This book will focus on recent developments with regard to these achievements. Antimicrobials that possess novel modes of action, particularly against drug-resistant organisms, so that they can be specifically targeted for clinical applications are required as a matter of urgency. In this regard, lantibiotics hold considerable potential as a consequence of their unusual structure, unique mechanisms of action, and their potency against multidrug-resistant bacteria. Today, close to 100 of these bioactive peptides have been described, the majority of which are produced by Gram-positive bacteria. The common feature that links all lantibiotics is the presence of a number of distinctive amino acids, which result from enzymatically mediated modifications after translation, including dehydration and cyclization, leading to the formation of eponymous (methyl)lanthionine bridges. These bridges convert the linear peptide chain into a polycyclic form giving structure and function to the peptide. It should be noted that only those peptides that display antimicrobial activity within the larger family of lanthionine-containing peptides or lanthipeptides are termed lantibiotics. Thus this book would be helpful in providing budding researchers and academicians with the information to understand recent trends in research in the developments of lantibiotics for combating various antibiotic and antimicrobial resistances. We are thankful to the Elsevier-Academic Press team: Stacy Masucci, Andre G. Wolff, Sam Young, Fahmida Sultana, and Greg Harris, for constant support from inception to the production of this book. We would also like to kindly acknowledge our Universities, for supporting such scholarly activities.

Chapter 1

Quandary of antibiotics and multidrug resistance development: a molecular genetics-based dilemma

Dipankar Ghosh¹∗, Paramjeet Singh¹#, Shubhangi Chaudhary¹#, Sampriti Sarkar¹# and Joseph Saoud²,    ¹¹Microbial Engineering and Algal Biotechnology Laboratory, Department of Biosciences, JIS University, Kolkata, West Bengal, India,    ²²Department of Natural Resources, McGill University, Montreal, QC, Canada∗, Corresponding author. e-mail address: [e-mail]

Abstract

Antibiotic resistant (AR) and multidrug resistance (MDR) are the most potent quandaries at present. AR and MDR raise the potential gainsay to the medical sectors with the possibility of precarious outbreaks and fear upon health management toward animal and human regimes. AR and MDR in microbial pathogenic communities cleverly rule out the efficacy of diverse ranges of antibiotics. Exposure to stress factors, microbial genetics, microbial physiology, and adoptive evolution are the most potential constraints in occurrence to AR and MDR phenomena in various pathogenic microbial communities. However, Innovation Gap upon novel antimicrobials discoveries boosts up rate of morbidity and mortality of AR and MDR pathogenic microbes. Hence, current literature summarization aims to emphasize on evolution and molecular mechanisms of antimicrobials with relevant experimental designs, molecular mechanisms of AR and MDR phenomena, and possible remedies to combat AR and MDR in corelation with microbial genetics, physiology, and impact of stress factors.

Keywords

Antibiotic resistance; multidrug resistance; antibiotics; microbial genetics; stress factors; adoptive evolution; pathogenic communities

Outline

Outline

Introduction 2

Brief history on assortment of antibiotics 4

Classification based on origin of antibiotics 4

Classification concerning the response for parasitic cells 5

Classification based on molecular mechanisms of antibiotics against the pathogen 5

General synopsis on antibiotic resistance and multidrug resistance phenomena 10

Molecular mechanism of antibiotic resistance and multidrug resistance phenomena 11

Impact of microbial genetics, microbial physiology, and stress factors on antibiotic and/or multidrug resistance 14

Technologies to address antibiotic resistance and/or multidrug resistance 16

Conclusion 18

Acknowledgment 19

References 19

Introduction

In the year of 1947, S. A. Waksman outlined the terminology Antibiotics as biochemical substances synthesized through the involvement of diverse microbial communities, which have the capacitance for stamping down the microbiological growth and demolishing bacteria and other groups of microorganisms (Mohr, 2016). The discovery of antibiotics with the purpose of curing or minimizing effectuate of life-threatening diseases is oneness successful inventions in 20th century. A combined effort of several industries and researchers has proved to be efficacious in such a way that in the 19th century only an inconsequential percentage of deaths have been occurred due to infectious disease as compared to deaths occur in the 18th century considering the consumption of antibiotics as a regular practice (Aminov, 2010). However, these antibiotics can be derived from animals, plants, tiny microorganisms, or from chemically synthesized molecules that can be mostly classified concerning origin (biochemical properties like chemical structures, surface charges, ionization features, availability of functional groups, molecular weight, solubility index, and pH stability), target of antibiotic specific to symptoms or diseases or specific part or organ systems of the body, and mode of actions (i.e., cell-wall biosynthesis suppression, cellular membrane disintegration, nucleic acid biogenesis curtailment, and alternation or suppression of protein synthesis) (Ghosh, 2016). Having a diverse chronological background, the discovery of antibiotics can be depicted into various phases: ancient era, preantibiotic era, early antibiotic era, and modern era (Hutchings, Truman, & Wilkinson, 2019). In ancient era (1700 BCE to CE 1100), it has been recorded that around 2000 years ago, that is, back in 1500 BCE, people in ancient civilizations, including Egypt, China, Serbia, Mesopotamia, Greece, and India, were using antibiotic-producing microbes to prevent deadly diseases. Mushrooms, beer yeast, and mold (whole cell or cellular extracts) have also been practiced to regale infected wounds. Around 1500 years old Eber’s papyrus (oldest medical document, preserved) includes the method of poultices with moldy bread to treat open wounds and also includes the uses of herbal drugs. Usage of compresses, tonics, and organic compounds by ancient physicians has also been reported. Recently, it was discovered that a methodology used about a 1000 years ago has the potentiality to kill MRSA (methicillin-resistant-Staphylococcus aureus) (Morar, Bhullar, Hughes, Junop, & Wright, 2009). Similar kinds of approaches have been seen evidently in other civilizations that can be presumed to be the oldest of approaches against diseases. During preantibiotic era (13th century to 18th century) the preliminary concept of antibiotics and their efficacy to fight against pathogenic agents are quite unknown. In the 16th century, communities have arrogated that few could channelize to salubrious individual descended with the same illness from a sick individual. The first systematic approach to chemotherapy has been reported in the 17th century when South American communities start using a powder that has made out of the Cinchona tree bark for treatment and prevention of malarial parasites (Gachelin, Garner, Ferroni, Tröhler, & Chalmers, 2017). A very limited diligence, which is cognized for the treatment against pathogens through the usage of energy-sapping bloodlettings, diets with herbs eradicated other diseases and their progressions. In the early 19th century, while the methods of culturing microorganisms develop, new horizons have been unveiled. One of the notable and prominent studies toward the development of antibiotics has invented by Surgeon Theodor Bill Roth who has performed experiments to investigate the role of bacteria in wounds and the cause of sepsis. It drives the antibiotic potentiality of the Penicillium sp. By that time, Sir Louis Pasteur has already described bacteria as contaminants. John Tyndall has conducted experiments to investigate contamination by pathogenic microbial regimes and, further, has been confirmed Bill Roth’s Theory. Moreover, Joseph Lister has proven this doctrine through proper experimentation the Bill Roth’s conceptualization (Rubin, 2007).

In later 19th century Robert Koch has looked into the correlational statistics betwixt an anorexic biocatalytic factor, that is, Bacillus anthracis and a unwellness Anthrax followed by the identification of anorexic biocatalytic factor of cholera, that is, Vibrio cholerae and tuberculosis, that is, Mycobacterium tuberculosis and has established the Koch’s Postulates, based upon diseases are classified as contagious and noncontagious. As a result of this classification, the methods of disinfection are formulated for the bar of the further spread of contagious diseases. After a comp literature survey, Jakob Henle has come to ratiocination upon infection; a particular infectious biomolecules can be transposed from an infected to healthy person. In early antibiotic era (19th century to early 20th century, i.e., 1900), the primary analytic thinking of antibiosis by bacterial population in soil habitat has been performed. The development of antiinfective drugs and the underlying concept of chemotherapy is widely credited to Paul Ehrlich, who developed the synthetic arsenic-based prodrug, salvarsan (salvation arsenic), and neo-salvarsan c. 100 years ago to treat syphilis having a causative agent Treponema pallidum (Williams, 2009). The biocatalytic efficacy of first synthetic antibiotic, the arsenic derivative arsphenamine (dioxy-diamino-arsenobenzol-dihydrochloride), with trade name Salvarsan, has been developed by Paul Ehrlich in 1909 as a joint venture of Alfred Bertheim and Sahachiro Hata (Mohr, 2016). Salvarsan is supplanted via the sulfonamide prodrug prontosil through the discovery of Gerhard Domagk (Gelpi, Gilbertson, & Tucker, 2015). In 1920, antibiosis by actinomycetes has also been familiarized. In 1928, Penicillin discovered an antibiotic or group of antibiotics generated naturally by certain blue molds. Penicillin becomes first antibiotic during the Second World War and subsequently is afterward sublimated by Norman Heatley, Howard Florey, and Ernst Chain and colleagues at Oxford. In 1930, sulfonamides were discovered, which were found to be veritably potential, as spacious–ambit antimicrobials that have still been used in clinical aspects at current days (Zaffiri, Gardner, & Toledo-Pereyra, 2012). In modern era (middle 20th century onward), antibiotics is describes as a biochemical compounds biosynthesize in microbes to destroy other pathogenic microbes by Waksman, whose work has been initiated in the blossom arena of antibiotics (i.e., 1940s–1960s). Most of the antibiotics discovered have been still in clinical use toward community services. Major groups of antibiotics have been descended from likely (1) Actinomycetes natural products (NP) (aminoglycosides, tetracyclines, amphenicols, macrolides, glycopeptides, tuberactinomycin, streptogramins, and cycloserine); (2) fungal NP (fusaric acid, cephalosporins, and enniatin); (3) bacterial NP (polymyxins and bacitracin) and some are synthetically produced antibiotics (nitrofurans, pyridinamines, azoles, phenazines, diaminopyrimidines, and ethambutol). However, the trend of discovering new innovative drugs has shown an innovation gap (1962–1980), which indirectly indicates that no major classes of drugs were developed in this period due to a lack of systematic approach, funding, and infrastructure. Synthetic biology has left major evidence of its innovative approaches by the discoveries of chloroquine in 1970 and mefloquine in 1980 using microbial communities as molecular cell factories, inclusive to other drugs (i.e., lipopeptides, pleuromutilin, oxazolidinones, and diarylquinoline) which are marketized at the final stage of the tenure of innovation gap (Ghosh, 2016).

According to the recent studies, there have been 45 new antibiotic candidates identified as prospective, having the potentiality for clinical exploitation for further clinical trials (Renwick, Brogan, & Mossialos, 2015). Out of the 45 new antibiotic candidates, 28 of those belong to the class of NP; on the other hand, 17 have a go at it to be synthetic, which comprises 12 classes, in which 7 have a bun in the oven to be new. In 1945, five new tetracyclines derived function for the first time as a new class of antibiotics and were introduced toward clinical application in 1948 (Chopra & Roberts, 2001). These comprised aminoglycoside, macrolides, pleuromutilin, polymyxins, distamycin, and fusidane, which have been trailed in phase III in the United States, but they have so soon been in use in other parts of the world for clinical purposes (Vijay et al., 2017). Ridinilazole and murepavadin are two new synthetic classes’ lies in Phase III clinical trials. Ridinilazole specifically targets Clostridium difficile by blocking cell division of the pathogen, the mechanisms of which are yet to be revealed (Vijay et al., 2017). Murepavadin blocks transport of lipopolysaccharide (LPS) to the outer membrane by inhibiting which has a novel mechanism of action, inhibiting LPS transport protein D (LptD) that bears a unique mode of biomolecular activity (Zaffiri et al., 2012). The historiographical trends of antibiotics have been an astounding locomote via the advancements on scientific inquiry and techniques, through different eras. In general the reason of death at the beginning of the 19th century strongly has tempted concerning the mass exploitation of diverse ranges of antibiotic candidates. The approaches of synthetic biology in this field are another add-on. Based on current scenario, the current chapter focuses to depict the trends of antibiotics classification considering different physicochemical parameters, precise molecular mode of action on pathogenic microbial communities, brief depiction on antibiotic resistance and multidrug resistance (MDR) phenomena, molecular mechanism of resistance upon antibiotic and multiple drug resistance phenomena, impact of microbial genetics, microbial physiology, and stress factors on antibiotic and/or MDR, and technologies to address antibiotic resistance and/or MDR.

Brief history on assortment of antibiotics

Following multiple physico-biochemical constraints, antibiotics can be grouped into different classes. The various groups are classed considering the origin of the antibiotic, its structure, the response it elicits against the parasitic cells, and how it acts against the pathogen through its’ molecular mechanisms.

Classification based on origin of antibiotics

Concerning origin, antibiotics can be broadly classified into two clades, that is, natural and synthetic antibiotics. Natural antibiotics are naturally biosynthesized via microbial communities as secondary metabolites through various naïve metabolic pathways. Since these compounds are secondary metabolites, thus they are not necessary for the microorganism to survive but are produced as and when it is required by the cell. Penicillin, gramicidin, chlortetracycline, and streptomycin have been considered few examples of the natural antibiotics generated by diverse ranges of microorganisms as secondary metabolites (Upmanyu & Malviya, 2020). These antibiotics are produced by the bacterial cells in competition to destroy and eliminate other bacterial cells in their close vicinity or microenvironment. The cellular debris thus are produced from killing of the cells is used up for nutrients for the bacterial survival that produce antibiotics. The large pool of natural antibiotics is obtained from bacteria that reside in the soil. The natural antibiotics are less severe and show lesser number of side effects in comparison to the synthetic antibiotics (Cesur & Demiröz., 2013). With the development of science over centuries, and with studies being conducted around the globe, the understanding of the structures and the mode of actions of the various antibiotics have diversified. This knowledge has enabled the biogenesis of antibiotic biomolecules chemically. In the lurch three decades, diverse categories of antibiotics are biosynthesized, and many are still underway in the laboratories around the world. Many of the synthesized antibiotics have been accepted and validated for use for treatment against various diseases. Some of the common examples of the synthetically produced antibiotics include cephalosporin C, 6-aminopenicillanic acid, linezolid, meropenem, and fluorocyclines. The synthetic antibiotics present a higher toxicity against the pathogens and have been found to work faster compared to the natural antibiotics (Fair & Tor, 2014; Pancu et al., 2021; Upmanyu & Malviya, 2020).

Classification concerning the response for parasitic cells

Concerning the response for parasitic cells, antibiotics can be broadly classified into major two clades, that is, bactericidal and bacteriostatic antibiotics. The bactericidal antibiotics are the ones that inhibit the growth of parasitic microorganisms by destroying and killing them by various mechanisms. Penicillins, cephalosporins, carbapenems, glycopeptides, and monobactams are some examples of bactericidal antibiotics (Etebu & Arikekpar, 2016; Pankey & Sabath, 2004). Bacteriostatic antibiotics that inhibit or restrict the bacterial growth instead of killing them as treatment against an infection fall under the class of bacteriostatic antibiotics. These antibiotics prevent the bacterial pathogens from dividing or reproducing. Current categories of antibiotics are not effective to regale certain diseases because, as the concentration of these antibiotics decreases, certain bacteria are able to grow again. Bacteriostatic antibiotics can also act as bactericidal for certain bacterial strains. Some examples of bacteriostatic antibiotics are minocycline, chloramphenicol, macrolides, trimethoprim, and sulfonamides (Pankey & Sabath, 2004; Peach, Bray, Winslow, Linington, & Linington, 2013).

Classification based on molecular mechanisms of antibiotics against the pathogen

Classification schemata for antibiotics based on the molecular mechanism against pathogenic microbial communities are quite diverse (Fig. 1.1), including β-lactam that inhibits the biogenesis of peptidoglycan layer of bacterial cellular wall; aminoglycosides and tetracycline that restrict biogenesis of polypeptides or proteins by acting on the 30S subunit of the ribosome and blocking it; quinolones that block the replication of DNA; and sulfa drugs that block the folate coenzyme biosynthetic pathway (Etebu & Arikekpar, 2016).

Figure 1.1 Synopsis of molecular mechanism of diverse ranges of antibiotics

. (A) Mode of action of beta-lactum group of antibiotics with PBPs to inhibit bacterial peptidoglycan layer biogenesis during cell-wall synthesis; (B) mode of action of polymyxin to inhibit bacterial cytoplasmic membrane integrity; (C) impact of rifampicin and sulfa drugs on nucleic acid biosynthesis including DNA and RNA; (D) impacts of chloramphenicol, erythromycin, tetracycline, and streptomycin towards bacterial protein biosynthesis inhibition). PBP, Penicillin-binding protein.

Beta-lactams and its mode of action in molecular level

Beta-lactams are antibiotics that affect and restrict the polypeptide biogenesis that are necessary for the synthesis of the cell wall in bacteria, which in turn inhibits the bacterial growth or kills them. The members of beta-lactam class of antibiotics consist of a highly reactive 3-carbon and 1-nitrogen ring. In short, there are certain enzymes associated with the bacterial cell called penicillin-binding protein (PBP) that accounts for the cross-linking of the peptide units during the peptidoglycan synthesis. The beta-lactam antibiotics have the capability to bind to the PBP enzymes, and, thus, in this process, they inhibit the cell-wall synthesis by interfering with the cross-linking of the peptidoglycan, which results in the cells to lyse and die. The most distinguished and luminary evidences of this clade of antibiotics are penicillins, cephalosporins, monobactams, and carbapenems (Adzitey, 2015; Etebu & Arikekpar, 2016).

Aminoglycosides and tetracyclines including mode of action in molecular level

The clades of antibiotic commodities participated in the forbiddance of 30S ribosome cover antibiotics families of aminocyclitol and tetracycline. Both the class of antibiotics, aminoglycosides which include kanamycin, streptomycin, framycetin, tobramycin, gentamicin, etc., and, the aminocyclitols which include spectinomycin follow a similar molecular mechanism of action against the pathogen by constipating at the 16S rRNA constituents of the 30S ribosomal fractional monetary unit. The spectomycins bind at the 30S ribosomal subunit and acts on the peptidyl-tRNA stability by inhibiting the translocation, which is catalyzed by the elongation factor without effecting any mistranslation of the protein (Etebu & Arikekpar, 2016; Upmanyu & Malviya, 2020). The interaction between the aminoglycosides and the 16S rRNA on the other hand inducts a conformity alteration in the composite that is sprang betwixt charged aminoacyl t-RNA on the ribosome and the m-RNA codon, which further results in the mismatching of the t-RNA causing mistranslation of the protein. This class of antibiotics comprising the aminoglycosides is the only class of antibiotics that show bactericidal response against the pathogen and are derived naturally. For instance, chloramphenicol along with the macrolides, azithromycin shows bactericidal response against Haemophilus influenzae, whereas chloramphenicol alone can destroy Streptococcus pneumoniae and Neisseria meningitides effectively. The variable regions of the highly conserved ribosomal proteins and RNAs show sequence differences among the various bacterial species, which forms the cause of the species-specific variation in the cell death mediated by ribosome inhibitors (Leach et al., 2007; Trimble, Mlynárčik, Kolář, & Hancock, 2016). The polymyxins are lipopeptides molecules of with a poly-cationic peptide ring and a short peptide attached to a hydrophobic fatty acid (Nation, Velkov, & Li, 2014; Vega & Caparon, 2012). The polymyxin peptides have bactericidal efficacy against mostly Gram-negative organisms and a few Gram-positive organisms (Vega & Caparon, 2012), although the modes of action of polymyxins are not fully understood but the primary studies show that polymyxins bind to the outermost cellular layer (i.e., outermost membranous layer) of the cells of Gram-ve bacteria and disrupt the LPS. The polypeptide portion of polymyxin binds to the negatively charged LPS, assisted by the interaction of the lipid tail with the fatty acids of the lipid-A moiety of the LPS. On adhering to the phosphorous residues of the lipid-A mediety, the magnesium and calcium ions that stabilize the membrane and crossbridge the adjacent lipid-A molecules get displaced by polymyxin that in turn destabilizes the outer membrane (Falagas, Kasiakou, & Saravolatz, 2005; Landman, Georgescu, Martin, & Quale, 2008). The displacement of these divalent cations makes the membrane weak, and the permeability barrier is disrupted; thus polymyxin subsequently allows several other drugs to pass through the membrane into the periplasmic space (Trimble et al., 2016).

Benjamin Duggar has identified tetracyclines from the soil bacterium having genus Streptomyces in the year 1945 (Etebu & Arikekpar, 2016). Chlortetracycline or aureomycin is the first member of the tetracycline class of antibiotics. The various members of this class of antibiotics have a four-ring structure of hydrocarbons to which a number of side chains are attached. The various members of this class are categorized into distinct generations on the basis of the method of synthesis (Etebu & Arikekpar, 2016; Upmanyu & Malviya, 2020). The first generation of tetracyclines includes chlortetracycline, tetracycline, demeclocycline, and oxy-tetracycline and is obtained by biosynthesis. The derivatives of semisynthesis comprise the second generation of tetracyclines, which include lymecycline, meclocycline, doxycycline, rolitetracycline, methacycline, and minocycline. Tigecycline is obtained from total synthesis and is member of the third generation of tetracyclines. This class of antibiotics demonstrates antimicrobial activity in the bacterial cells by acting on the ribosomes similar to the aminoglycosides. The various tetracyclines, such as chlortetracycline, doxycycline, tetracycline and minocycline, combine with 30S subunit of the ribosome by acting on the conserved sequences of the 16S r-RNA that forecloses the intractability of the t-RNA to ribosomal complex associated A-site. This suppresses polypeptide biogenesis by hindering the addition of amino acids to the polypeptide chains in the bacterial ribosome (Sánchez, Rogers, & Sheridan, 2004).

Macrolides and its mode of action in molecular level

Macrolides are categorized by having 14–16-membered macrocyclic rings of lactose with different deoxy sugars D-desoamine and L-cladinose attached to it. The metabolic products of a soil-inhabiting fungus Saccharopolyspora erythraea was isolated and studied in 1952 by J.M. McGuire, which was then identified as the first antibiotic in the class of macrolides (Hong, Zeng, & Xie, 2014; Tenson, Lovmar, & Ehrenberg, 2003; Upmanyu & Malviya, 2020; Vakulenko & Mobashery, 2003). Macrolides are often given to patients who are allergic to penicillin and compared to penicillin; these antibiotics have a wider spectrum activity (Etebu & Arikekpar, 2016) Macrolides can either show bactericidal activity by killing the microorganisms or act as bacteriostatic antibiotic and inhibit the growth of the microorganism by effectually inhibiting the synthesis of proteins in the bacterial cells. The inhibition of the protein synthesis is brought about by macrolides by their binding to the ribosome of bacterial cell and in turn preventing the addition of amino acids to the polypeptide chain during the synthesis of the protein. The primary targets of the macrolides are the 50S ribosomal subunit that stimulates the dissociation of the peptidyl-tRNA molecule from the ribosomes during the elongation process of protein synthesis (Kong, Schneper, & Mathee, 2010). Some of the examples of macrolides include azithromycin, clarithromycin, and erythromycin.

Quinolones and its mode of action in molecular level

Scientists in the search of antimalarial drugs discovered a compound called nalidixic acid that was the first ever member of this class of antibiotics. The compound was identified during the process of generation of quinine in the sixties as an impurity (Aldred, Kerns, & Osheroff, 2014; Bronson & Barrett, 2001; Fàbrega, Madurga, Giralt, & Vila, 2008). These compounds have the capability to interfere with the replication and transcription process of DNA in bacterial cells. The basic molecule was used for the development of two significantly paramount groups of compounds: naphthyridones and quinolones that comprise norfloxacin, cinoxacin, ciproxacin, ofloxacin, nalidixic acid, enoxacin, sparfloxacin, temafloxacin, and so on (Choquet-Kastylevsky, Vial, & Descotes, 2002). Quinolones generally have a two-ringed structure, but in the recent developments, the newer generations of these antibiotics have an added ring structure that has allowed these compounds to exhibit an extended spectrum of antimicrobial activity to some bacterial species, particularly against the quinolones-resistant anaerobic bacteria. Most of the bacterial species encode for two distinct type II topoisomerases that include gyrase and topoisomerase IV (topo IV). Topoisomerase catalyzes and modulates the supercoiling of the chromosomes, which involves breakage and rejoining reactions that is found to be essential in the synthesis of DNA, transcription of mRNA, and cell division process. Antibiotics in the class of quinolones target the reactions of breakage and rejoining and, thus, target the DNA-topoisomerase complexes in the bacterial cell. They also target the DNA gyrase or topoisomerase II and topoisomerase IV and interfere with the maintenance of the topology of the chromosome. The quinolones prevent the rejoining of the strands by trapping these enzymes at the stage where the DNA is cleaved. The antibiotic forms a very stable complex with the interaction between the drug-bound topoisomerase and the DNA that is cleaved. This ability of the quinolones aids in the killing of the bacterial cell. The final result of the activity of quinolones on the bacterial genome is the creation of double-stranded breaks in the DNA (Aldred et al., 2014; Fàbrega et al., 2008). These DNA breaks are trapped in this condition by the covalent interaction between the topoisomerase and the antibiotic compound bound to it. The quinolones–topoisomerase–DNA complex remains intact, and, thus, the replication forks are blocked, resulting in the stoppage of the progression of the replication machinery of the DNA. This has an overall impact on the synthesis of DNA, which exerts a bacteriostatic effect on the bacterial pathogen and causing cell death (Brunton et al., 2015; Choquet-Kastylevsky et al., 2002; Etebu & Arikekpar, 2016).

Sulfonamides and its mode of action in molecular level

The first groups of antibiotics used in therapeutics are sulfonamides and are still an essential part of medicine and veterinary practice. Sulfonamides are used to treat a wide spectrum of infections including septicemia, tonsillitis, meningitis, meningococcal, urinary tract infections, and bacillary dysentery, inhibiting both Gram-positive as well as Gram-negative bacteria, including Escherichia coli, Nocardia, Salmonella, Klebsiella, Enterobacter sp. and Shigella sp., Chlamydia trachomatis, and some protozoans. Studies on sulfonamides have shown that they also have the ability to hinder and obstruct cancerous cell agents (Choquet-Kastylevsky et al., 2002; Etebu & Arikekpar, 2016). The originally developed sulfonamides are synthetically produced with antimicrobial activity and comprised sulfonamide group. These antibiotics are generally bacteriostatic, but they become bactericidal when the concentration is adequately high or other environmental factors that are unfavorable for the bacterial cell are accompanying the presence of the antibiotic. The unfavorable environmental conditions include adverse temperatures, the presence of antibodies against the pathogen, the lack of availability of nutrients required by the pathogen, and the presence of toxic and proteolytic products (Shakoor, Ayub, & Ayub, 2013; Upmanyu & Malviya, 2020). The mechanism of action of sulfonamides involves the inhibition of synthesis of folic acids in the bacterial cells, which in turn inhibits the synthesis of the purines and pyrimidines that act as the building blocks for DNA. Bacterial cells produce an intermediate, para-aminobenzoic acid (PABA) during the synthesis of folic acids. Sulfonamides are found to be structurally analogous to PABA. PABA reacts with pteridine in the first step of synthesis of folic acid in the presence of the enzyme dihydropterate synthase. The reaction produces dihydropteric acid that further reacts with glutamic acid, which produces dihydrofolic acid. This reaction is catalyzed by the enzyme dihydrofolate synthase. Tetrahydrofolic acid is formed by the further conversion of dihydrofolic acid by the enzyme dihydrofolate reductase (Shakoor et al., 2013). This tetrahydrofolic acid is employed through the bacteria for alleviating nonprotein nitrogenous bases, that is, purines and pyrimidines yields. In the comportment of sulfonamides, the dihydropterate synthase enzyme binds to the sulfonamide with a higher affinity for the antibiotic and, thus, blocking the synthesis of folic acid. Moreover, sulfonamides, on being incorporated into the precursors, results in the formation of pseudometabolites that show antibacterial activity and are highly reactive (Beaber, Hochhut, & Waldor, 2003; Choquet-Kastylevsky et al., 2002).

General synopsis on antibiotic resistance and multidrug resistance phenomena

Antibiotic resistance is basically the ability of pathogenic microorganisms to conquer the medications that are used to eradicate them, via cellular recognition. The spread of antibiotic-resistant bacteria is an emerging public health issue that causes a large number of complexities in treatment and prevention of infectious and widespread diseases caused by tiny microorganisms causing life-threatening diseases like tuberculosis, pneumonia, HIV, influenza, malaria, cystitis, cancer, and candidiasis (Aibuedefe Osagie, 2019). The development of the antibiotic resistance in microorganisms is mostly due to an inappropriate and unnecessary use of antibiotics. The resistant microorganisms have developed over years, and as a result, several difficulties, regarding the treatment of infections emerged with the resistant microorganisms, are being faced. Though this is a problem, worldwide but particularly shocking in the countries like India due to large population, poor economical state, imperfect infrastructure, inconsistent health measures, and the overavailability of unprescribed antimicrobial drugs in pharmaceutical stores. These kinds of negligence are working as acceleration factor to the emergence of drug resistance in India (Ghosh, 2016). Assorted hereditary mechanics of genetics and modifications pertained to resistance aspect upon exposure of antibiotics have been identified, which can also been classified into four major classes, namely, acquired and instinctive resistance involves genetic alterations by quarry alteration, efflux system, and brought down permeableness. For example, as vancomycin does not pass in the outer membrane so Gram-negative bacteria is naturally resistant to vancomycin. Similarly, L-form shape of bacteria that are wall-less forms of the bacteria, and the bacteria, such as cell wall-less cell Mycoplasma and Urea plasma, are naturally resistant to beta-lactam antibiotics that inhibit the cell-wall synthesis (Cesur & Demiröz., 2013); acquired resistance involves mutation horizontal gene transfer (HGT) via chromosomal or extra chromosomal structures like, transposons and integrons, plasmids that can acquire and express new genes; cross-resistance includes a resistance, developed against all the antibiotics belonging to the same class with the help of a single mechanism. For example, the cross-resistance of Burkholderia cepacia to SDS, beta-lactams, kanamycin, erythromycin, novobiocin, and ofloxacin due to the di-sulfide bond formation system; MDR of specific species of microorganism to at least one antimicrobial drug in three or more antimicrobial categories. For example (1) vancomycin-resistant Enterococci; (2) MRSA; (3) extended-spectrum β-lactamase producing Gram-negative bacteria; (4) multidrug-resistant Gram-negative rods (MDR GNR) MDRGNR bacteria, such as Enterobacter species, E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa; and (5) multidrug-resistant tuberculosis (Sid Ahmed et al., 2019). The emersion of resistance upon multidrug applications during last few decades has resulted in the increment microbial infections significantly. Since the introduction of penicillin during the 1930s, in early antibiotic era, a number of antimicrobial agents have been developed and packetized for general clinical use that helped minimizing the rate of human mortality that are previously associated with microbial infections before these inhibitory agents came into field. Initially it raised optimism about the possibility of the successful treatment of all microbial infections but the concept got disabused when emergence of drug resistance reported. The development of MDR microbes with higher infectivity rate and transmissibility, increased virulence, and enhanced morbidity occurred due to multiple mutations enhancing high levels of resistance to the antibiotic classes available for the treatment (Périchon, Courvalin, & Stratton, 2015). However, MDR is a significant threat to public health and hence there’s an urgent need to understand the molecular mechanism of antibiotic resistance and MDR phenomena and to develop tools that predict the potential for the evolution of resistance to novel drugs and treatment methods (Jacopin, Lehtinen, Débarre, & Blanquart, 2020).

Molecular mechanism of antibiotic resistance and multidrug resistance phenomena

Antimicrobial resistance (AMR) has been known since the ancient times and is believed to be a consequence of the interactions between various microorganisms and their environment (Ray, Das, & Suar, 2017). This section discusses the genetics and molecular mechanisms involved in the development of MDR (Fig. 1.2). There are two mechanisms by which bacterial cells can attain resistance to antibiotics—intrinsic and acquired mechanisms. The naturally occurring genes found on the chromosome of the host, such as, the various multidrug-resistant efflux systems and the AmpC β-lactamase of Gram-negative bacteria, specify the intrinsic mechanisms for antibiotic resistance. The acquired mechanism for antibiotic resistance is attributed to the mutations in genes that are targeted by the antibiotic and also by the transfer of resistance determining factors carried by plasmids, transposons, bacteriophage, and other mobile genetic materials (Munita & Arias, 2001; Ray et al., 2017). Generally, the transfer mechanisms of genetic materials followed include transformation by the incorporation of the genetic material, such as plasmids and other DNAs from dead cells in the chromosomal DNA; conjugation of plasmids and conjugative transposons; and transduction with bacteriophage. It has been observed as a regular phenomenon of exchange of genes among organisms of the same genus, but this phenomenon has also been reported to occur among different genera, which includes the transfer of genetic materials between Gram-positive and Gram-negative bacteria that are found to be evolutionary very distant to one another (Munita & Arias, 2001). Plasmids are self-replicating, extra chromosomal DNA, which can be distinguished from the chromosomal DNA from their origins of replication and contain genes that code for resistance against antibiotics and several other characteristic properties. A single bacterium can contain a number of plasmids, and the genes of all these plasmids make up the total genetics of the bacterium (Wright, 2011). Transposons on the other end are genetic elements that are very mobile and can be present on a plasmid or they can integrate in another transposon or the chromosome of the host. The transposable elements in general are a piece of DNA that contain regions on the terminal ends that take part in the recombination process and encode for proteins, such as transposase or recombinase that aids in the recombination and incorporation of the transposon from a region of DNA to another region within the same DNA or another DNA present in the bacterial cell. Conjugative transposons have features that are similar to plasmids and have the unique ability of facilitating in transferring endogenous plasmids from one organism to another (Ashraf, Mustafa, Rehman, Khalid Bashir, & Adnan Ashraf, 2019; Wright, 2011). A collection of genes or a gene cassette is present in an integron and is classified generally based on the integrase protein sequence that imparts the function of recombination. Integrons have the ability of integrating in a very stable manner into regions of DNAs where they are delivered. In a single exchange, integrons are able to deliver multiple new genes to the DNA they incorporate into, in particular genes related to resistance to antibiotic drugs (Wright, 2011). Soon after Alexander Fleming discovered the very first antibiotic, penicillin, he reported the existence of microorganisms resistant to the antibiotic. It was unpredictable how quickly and rapidly bacterial populations would develop the ability to combat various antibiotic drugs and be able to elicit MDR through various mechanisms. Initially, plasmids known as resistance factors or R factors, which specified a collection of antibiotic resistance determinants against individual antibiotics, are thought to be the vehicles for the rapid spreading of antibiotic resistance features among bacterial populations (Kapoor, Saigal, & Elongavan, 2017). Various mechanisms have been identified and studied for the development and expression of antibiotic resistance since the time antibiotics have been introduced for clinical treatment. We are nearing 100 years to the discovery of the very first antibiotic by Fleming, and scientists are yet to identify an antibiotic that can evade the development of antibiotic resistance in microorganisms (Alekshun & Levy, 2007; Kapoor et al., 2017). There are several ways out that have been identified to escape the molecular action of diverse ranges on antibiotic in broad spectrum of microbial regimes, including antibacterial drug degradation by bacterial enzymes, reduction in permeability to the drug by bacterial cells, escalated efflux mechanism of drug resistance, alteration of drug target with microbial regimes, and alternative pathways upregulation within microbial regimes to escape antibiotics

Figure 1.2 Plethora of antimicrobial resistance and multidrug resistance in pathogens.

The current figure deals with molecular changes in specific targets in pathogens and inactivation of antibiotics to neutralize antimicrobial’s efficacies to kill or inhibit pathogens.

Many bacteria produce enzymes known as beta-lactamase that have the capability to degrade the beta-lactam class of antibiotics. The beta-lactamase enzymes are able to do so by acting upon the beta-lactam ring of the molecules and breaks down the antibiotic. The degradation of other class of antibiotics is also achieved by bacterial populations by various mechanisms. For instance, certain antibiotics, for example, the chloramphenicol molecules, can be degraded by the addition of specific groups to the molecule. Chloramphenicol molecules can easily be acetylated by the addition of an acetyl-CoA group to the hydroxyl group of the drug. The addition of the group is catalyzed by the enzyme, acetyl transferases. Furthermore, the enzymes, phosphotransferases, acetyl transferases, and adenylyl transferases cause the addition of phosphate, acetyl CoA, and adenylyl groups, respectively, to aminoglycoside class of antibiotics for their degradation (Tanwar, Das, Fatima, & Hameed, 2014). The reduction in permeability of antibiotics can greatly impact the affectivity of the drug against the pathogen as it prevents it from accessing the cell of the bacteria and, thus, cannot bind to the target (Pelfrene, Botgros, & Cavaleri, 2021). This mechanism of resistance against antibiotics is seen in Gram-negative bacteria as they possess porins in their outer membrane, which are exceedingly small in size. This poses a major barrier to the permeability to a vast number of antibiotic molecules, not allowing them to impact the bacterial cell. Moreover, Mycobacterium sp. also follows a similar mechanism of reduced permeability to the antibiotics as they comprise a thick layer of mycolic acid that is a waxy material, which does not allow a majority of drugs to enter the bacterium (Kapoor et al., 2017; Tanwar et al., 2014). A diverse number of bacterial species have developed more effective mechanisms to resist antibiotic drugs by possessing efflux pumps (Nikaido, 2009). These efflux pumps are able to pump out the antibiotic drugs actively out of the cells of the bacteria. The result of pumping of the drug out of the cells is that it reduces the concentration of the antibiotics to a concentration lower than at which it can have a bactericidal effect on the cell. This mechanism has been developed by bacterial cells to possess resistance to a vast spectrum of antibiotics, including aminoglycosides, tetracyclines, and sulfa drugs (Pelfrene et al., 2021). S. aureus, E. coli, and Pseudomonas sp. are examples of bacteria that have emerged resistant against antibiotics by adopting the mechanism that uses efflux pumps (Nikaido, 2009). A variety of antibiotics initiate their mechanism of action by binding to a specific site within the cells of the bacteria so as to bring about its bactericidal or bacteriostatic activity against the bacterium (Över, Gür, Ünal, & Miller, 2001). A modification in the target site of the drug hinders with the activity of the molecule, catering to the development of resistance by the bacterial cells against the antibiotic. The development of such antibiotic and MDR is attributed against phenolics like chloramphenicol and macrolides. These drugs show drastic reduction in their antibiotic activity and get inhibited by a modification in the ribosomal binding sites (Leclercq, 2002; Över et al., 2001; Schwarz, Kehrenberg, Doublet, & Cloeckaert, 2004). A few of the bacterial populations adapt to develop an alternative pathway to the pathway that is blocked or affected by the action of particular antibiotics. A very common example is the utilization of folic acids by bacterial cells, which are preformed instead of them being synthesized by the bacterium itself. This mechanism hinders with the activity of sulfonamides as they act on the folic acid synthesis pathway to exhibit their antibacterial activity by competitive antagonism (Fair & Tor, 2014; Fernández-Villa & Rojo, 2019; Tanwar et al., 2014).

Impact of microbial genetics, microbial physiology, and stress factors on antibiotic and/or multidrug resistance

Microbial genetics encompasses the study of a variety of information inheritance mechanisms in microorganisms like the bacteria, archaea, viruses and some protozoa, fungi, and more. These studies over the course of time prove that along with genetics, factors as microbial physiology and involved stress have a significantly apparent impact upon the antibiotic and MDR that has been seen rapidly increasing in the given times and has been cited as a major problem for the current times due to factors as an intensive use of antibiotics from agriculture to medicine (Ghosh, Saran, & Saha, 2020; Levy & Marshall, 2004; Ben Maamar et al., 2020) and the lack of discovery of newer antibiotics that could possibly be used in its stead to bring the situation under control. AMR generally stems from either modification of the antibiotic, modification or reduction in access to the antibiotic target, and others. Resistance determinants hence can be the result of mutations in the cellular genes, gene acquisition, or the mutation of these acquired genes. Furthermore, the ability of pathogenic microorganism to acquire, concentrate, and disseminate resistance genes is hugely determined by a variety of genetic elements, including plasmids, transposons, bacteriophage, integrons, and more (John & Rice, 2000). Microbial genetics comprises a wide range of things; later we’ll discuss a few factors that have been primarily studied and the conclusions that have been drawn. Gene amplification essentially refers to the increase in the number of copies of a gene sequence in an organism. It is not usual since it is not proportional to the increase of surrounding genetic content. Gene amplification holds much importance in various factors as presenting characteristics, phenotypic characters are determined by it, it affects disease, sometimes contributing to its cause, causes diversity, makes one susceptible to variety of factors, cause resistance, affect adaptation, and much more. The field of genetics holds much importance when it comes to the treatment of diseases and medicine. Resistance here presents itself as a problem since it hinders in the treatment of a disease, rendering the process ineffective compromising the system of effective healthcare. Microorganisms in the presence of any detrimental factor adapt to survive and genetic amplification hence serves to facilitate it and, thereby, aids in the development of resistance, the more the exposure, the added stress, the more the adaptation. Like bacteria can adapt to toxic levels of antibiotics using regulatory mechanisms that such as mutation or HGT, which in turn might lead to the degradation or sequestration of the antibiotic, hinder its uptake, its exclusion from the cell via efflux or prevention of binding to the target molecule, and much more (Aibuedefe Osagie, 2019; Levy & Marshall, 2004; Sandegren & Andersson, 2009). There are certain evidences that show antibiotics can induce mutagenesis within bacterial communities, which is called antibiotic-induced mutagenesis (AIM). Bacteria have been found to have mechanisms that promote genetic variation when subjected to specific conditions (Aruković, Fetahović, & Pehlivanović, 2020). AIM occurs due to the development of single-stranded DNA, and consequent activation of the SOS response that happens to be a reply to DNA injury in which mutagenesis and DNA repair are induced. Quinolones have a mutagenic effect on bacteria and are amongst the extensively used family of DNA damaging antibiotics in use, whereas B-Lactams happen to be the most widely used antibiotics in clinical medicine. It has been noted by numerous studies that subinhibitory concentrations of quinolones raise the frequency of resistance mutants in some bacteria. However, β-lactams have been demonstrated as a potential triggering agent for the activation of SOS response in bacteria (Aibuedefe Osagie, 2019; Aruković et al., 2020; Nikaido, 2009). In the case of antibiotic-induced recombination and lateral gene transfer phenomenon, mutagenesis and recombination happen to be largely responsible for evident variability amongst microorganism, but there are various other factors that make recombination more suitable to occur. These include physical proximity, more likely to exist between members of the same community, for the structuring and geographical location greatly impacts the microbial community evident in the region (Aibuedefe Osagie, 2019; Aruković et al., 2020). The environmental factors play a great role too. HGT can occur via modes of transformation, transduction, and conjugation. In addition, efflux pump–associated MDR depicts that the efflux of drugs plays a potent persona upon drug resistance angle, that is, tetracycline. Major facilitator superfamily is amongst the heaviest taxa of transporters and carries panoptic array of efflux pumps (Aruković et al., 2020; Nikaido, 2009; Bhardwaj & Mohanty, 2012; Chitsaz & Brown, 2017; Dashtbani-Roozbehani & Brown, 2021). Plasmid-borne amplifications happen to be among the first studied examples of an increased level of antibiotic resistance resulting from increased gene copy number in early 1970s; different R plasmids are discovered to increase in size when grown in the presence of antibiotics. The first example was plasmid Nr1 (also called r100) (Bennett, 2008). Chromosomal gene amplifications (Normark & Normark, 2002) have also been involved toward genetic amplification that led to increased antibiotic resistance using an E. coli strain containing a promoter mutation for the high production of the AmpC β-lactamase; they further studied higher levels of resistance by growing the cells in increasing concentrations of ampicillin (Nikaido, 2009; Sandegren & Andersson, 2009). Microbial physiology also take part in the antibiotic susceptibility of bacterial cells happens to be impacted by their physiological states and influence the development of MDR. Biofilms have been found to be provided more antibiotic resistance. However, primary trial to comprehend it, few studies have been performed on certain model biofilm-forming microbes following limiting diffusional barrier of chemotherapeutic compounds via biofilm-forming microbial communities. Nutritional stress and anaerobic conditions for aerobic cells also causes the development of biofilms (Dincer, Masume Uslu, & Delik, 2020; Poole, 2012). However, it has also been added that this mechanism cannot produce large or extensive resistance in the microbes. There also have datasets those link antibiotic resistance to biofilm formation in P. aeruginosa biofilms via the production of periplasmic β-(1–3)-glucans (Nikaido, 2009; Pang, Raudonis, Glick, Lin, & Cheng, 2019) and an efflux system. Persister population have been found to limit the efficacy of administered antibiotic and happen to be a type of survival strategy in bacteria where mixtures of phenotypically different populations of bacteria are produced naturally so that a part of this population can thrive in adverse situations. It has also been studied that the elevated quantity of antibiotics does not essentially destroy entire bacterial classes among these cells, and a persister population thrives, which happens to be genetically identical with the susceptible cells (Frieri, Kumar, & Boutin, 2017; Nikaido, 2009). Factors other than antimicrobial selective pressure exist and also affect the emergence and persistence of resistance determinants (Levy & Marshall, 2004). Quorum sensing is cell-to-cell communication amongst bacterial cells at the molecular level controlled by chemical signaling molecules-auto inducers. In bacteria, it helps recognize the population density by measuring the accumulation of signaling molecules secreted from members of the community (Dincer et al., 2020). The other stress factors include nutritional stress, oxidative stress, pH-temperature-induced stress issues. Nutritional stress includes the reduced metabolic activity, absence of oxygen, and nonoptimal growth conditions that give rise to resistance amongst the organisms, as in P. aeruginosa (Poole, 2012). During oxidative stress in microorganisms growing aerobically, oxidative stress is evident in the form of reactive oxygen species, which destroy subcellular parts and hence minimize associated adoptive receptions betrothed to aid endurance in the mien of such conditions (Poole, 2012). Heat-pH-induced stress including immune defenses is also considered a major role player in this scenario (John & Rice, 2000). Growth-retarding accentuate factors include nutrient famishment, lower pH, raised osmotic pressure, hypoxia, extreme temperature shifts, or antimicrobial abandonment, as described earlier, and these cause the bacteria to actuate a stress-induced acceleration in the rate of microbial mutation, which is also termed adaptive mutagenesis, and these activate the SOS responses that typically follow DNA damage (Foster, 2007; Poole, 2012). Based on these studies and supposed conclusions, the impact of utilization and marketization and the associated chain of events that unfold with respect to the microbial communities and the spread of antibiotic-resistant genes (ARGs), in addition to their potential direct and indirect effects on human health, should be gravely discussed. To provide a better and more accurate risk assessment related to ARGs for human health, the actual transfer ability of mobile ARGs should be identified through various approaches and studied in detail (Ben Maamar et al.,