eChapters

Introduction to Antibiotic Discovery

Antibiotics are the drugs that are used to treat illnesses caused by bacteria in humans and animals. These act by killing or inhibiting the growth and multiplication of bacteria. Antibiotics were considered as “magic bullets” that selectively kills microbes responsible for disease causation, but at the same time would not affect the host (Zaman et al., 2017).

Louis Pasteur in 1877, first discovered the properties of antibacterial compounds with the inhibition of anthrax by saprophytic bacteria. In 1928, Alexander Fleming observed that the growth of Staphylococcus aureus in petri dishes was inhibited by substances produced by the fungus Penicillium chrysogenum, which led to the discovery of the first antibiotic, penicillin (Aminov, 2017). Selman Waksman, discovered multiple antibiotics from filamentous actinomycetes living in the soil and defined an antibiotic as “a compound made by a microbe to destroy other microbes”. Following the discovery of Penicillin, various antimicrobial compounds such as sulphonamides, aminoglycosides, tetracyclines, lipopeptides, oxazolidinones, glycopeptides, streptogramins, and quinolones were discovered in the “Golden era” of antibiotics during the period from the 1940s to the 1960s and most of the antibiotics still in current use were discovered during that period (Salam et al., 2023). Millions of metric tons of newer classes of antibiotics have been produced in last 60 years since its inception.

Introduction

Measurement of pH of a solution and preparation of different types/strength of buffers are common practices in a laboratory, particularly in the Biochemistry and Molecular Biology laboratories. Therefore, in this chapter you will be acquainted with the procedures of measuring pH of a solution as well as preparation of some of the commonly used buffers in a laboratory.

Potential or Power of Hydrogen Ion (pH)

Measurement of pH of a solution is measuring the molar concentration of hydrogen ion [H+] in the solution, which is an indication of the acidity or basicity of the solution. The symbol pH stands for “power of hydrogen” and numerical value for pH is just the negative logarithm of the molar concentration of hydrogen ions [H+]. The ionic product of water (Kw ) is the basis for the pH scale. The total hydrogen ion concentration from all the sources and it can be experimentally measured.

The pH scale designates the concentration of H+ (and thus of OH−) in an aqueous solution, which ranges between 1.0 M H+ and 1.0 M OH−. The term pH is defined by the expression

Thus, the ionic product of water makes it possible to calculate the concentration of H+, with given concentration of OH−, and vice versa. The value of 7 for pH of a neutral solution is not an arbitrarily chosen figure; it is derived from the absolute value of the ionic product of water. A solution having pH greater than 7 is basic, and the concentration of OH− is greater than that of H+. A solution having a pH less than 7 is acidic.

Introduction

Different types of culture media are used for culturing microbes and plant tissues. Preparing as well as sterilizing them properly following appropriate method with safety for the desired purpose are important parts of research work. In this chapter, we will discuss briefly about different types of media, their composition, preparation and sterilization in laboratory for microbial or plant tissue culture.

Culture Medium

Ingredients mixed to prepare solid, liquid or semi-solid substance to support the growth of microorganism or plant tissue in vitro (outside the body) is called culture medium. Frequently, it becomes important to grow microorganism or plant/animal tissue in vitro for any or all of the following purposes:

• To study characteristic features or properties of microorganisms.

• To identify the causal organism(s) of infection in a clinical sample, so that appropriate remedy can be determined.

• To culture/multiply microorganism or plant/animal tissues in vitro for research or commercial purposes.

• To prepare/produce biological molecules, vaccines, antigens, proteins, metabolites, etc. in vitro for research or industrial purposes.

Introduction

Flowering plants (Angiosperms) are identified by their morphological characteristics like flower, fruit and seeds that help their taxonomic characterization into different species, genera, families and orders. The floral characters which are used for taxonomic characterization of plants include the type of inflorescences, perianth structure, floral symmetry, arrangement of floral leaves, type of androecium, stamens, gynoecium, carpel, ovule, etc. The type of fruit and seed also play important roles in taxonomic classification. In this chapter we will briefly discuss about various morphological features of flowering plants (in botanical terms) that are useful in taxonomy.

Nomenclature of Plant

Nomenclature of plant is the formal and scientific naming of plants. It is related to but distinct from taxonomy for grouping and classifying plants and provide botanical name.

The scientific/botanical nomenclature started with Species Plantarum by Carolus Linnaeus in 1753. Botanical nomenclature is governed by the International Code of Nomenclature for algae, fungi, and plants (ICN), which replaces the International Code of Botanical Nomenclature (ICBN).

ICBN and ICN

International Code of Botanical Nomenclature, (ICBN) was adopted by the VIIth International Botanical Congress held in Stockholm, July1950. While the International Code of Nomenclature for algae, fungi, and plants (ICN) was adopted by the XVIIIth International Botanical Congress held in Melbourne, Australia in July 2011.

The response of any living cell to a xenobiotic (including antibacterial agent) is significantly affected by the prevailing local environmental conditions. In case of bacterial infection, it may include the presence of other microbes, no matter commensal or pathogenic, or biochemical alterations at that particular site of bacterial infection in host. All these host and bacterial factors play a critical role in the evolutionary responses of bacteria but usually go unconsidered while determining the microbial sensitivity to a particular antibiotic in a clinical setting. These also include the direct and indirect interspecies interactions that can affect the responses of individual bacterial species and surviving microbial communities to a particular antibiotic therapy. These interactions may significantly alter the qualitative and quantitative responses, potential phenotypic changes and the final trajectory of evolution of antimicrobial resistance. Due acknowledgement and plausible interpretation to these interactions may help to reduce the upsurge in AMR and its associated morbidity and fatality and pave path to more proficient clinical success.

The factors participating in drug- microbe interaction can be classified into two broad groups: host factors and microbial factors. The environmental milieu of the interaction is chiefly regulated by the gut microbiota with stressors causing deflections. The gastrointestinal bacteria amend host mental and physiological cadence via microbial metabolites such as butyrate, polyphenolic antioxidants, vitamins, and amines, affecting the overall ready perception and active rebuke to a stimulus. Lifestyle stressors such as fluctuating sleep and erratic eating patterns (western diet) disturb the host circadian system along with the gut microbiome. The overall impact is impaired detoxification (decreased conjugation of bile acids or increased production of hydrogen sulphide), substrate oxidation and energy regulation (reduced butyrate production) in the host affecting the overall health and productivity.

Introduction

We come across a variety of chemicals every day in a laboratory. While some of them are relatively safer, others are extremely hazardous. Thus, it is very important to know the potential hazards that can be caused by the chemicals present in the laboratory and the safety measures needed to be followed to avoid the hazards. Additionally, we should also be aware of the steps necessary to be taken on such hazardous situations. According to IUPAC, a hazardous chemical mean the chemical which has the potential to cause damage in various ways. In a laboratory, there are chemicals that might be irritant, carcinogen, respiratory fibrogen, systemic poison, asphyxiant, flammable, etc. which have the potential to cause hazard to the workers or environment. Therefore, it is important to use appropriate procedure to reduce the risk and protect the health/safety of laboratory personnel, the public and the environment.

Types of Hazards Associated with Chemicals

Chemical hazards: The hazards caused by chemicals can be broadly classified into three categories.

1. Health hazard: This is also known as toxic effects of chemical. The risk associated with a chemical in laboratory depends on the extent of exposure and the inherent toxicity of the chemical. Many chemical reactions form the product that might be much more toxic than the reacting chemicals. For example, inadvertent mixing of formaldehyde (a common tissue fixative) and hydrogen chloride results in the generation of bis(chloromethyl) ether, a potent human carcinogen. For most of the chemicals, the route of exposure (through skin, eyes, gastrointestinal tract, or respiratory tract) is more important (Figure 17.1) that must be considered during risk assessment.

Introduction

Proper electrical wiring and earthing are crucial for laboratory safety and functionality. A laboratory houses several installations/equipment that demand stable/continuous power supply. Inappropriate or faulty wiring and earthing may lead to significant risks, including electrical shocks, damage to equipment, and even incidence of fire. Therefore, understanding the requirements of electrical wiring and earthing is essential for designing, construction, and maintenance of laboratory. This chapter aims to provide a basic knowledge of electrical wiring and earthing necessary for a laboratory. Importance of earthing in mitigating electrical hazards and ensuring integrity of electrical installations are also described.

Basics of Electrical Wiring

Basic understanding of electrical wiring is necessary for laboratory safety and efficient electrical functioning. The potential difference between two electrical points (negative and positive) is known as Voltage, which drives the flow of electric current. This is measured as volt (V). The rate of flow of electricity in a circuit is known as Current (I), which is measured as ampere (A). Current can be either direct (DC) or alternating (AC). Opposition/resistance experienced in the flow of current in circuit is known as Resistance (R) and it is measured in ohms (Ω). Higher the resistance means lesser the current flows at a given voltage. The rate at which electrical energy is transferred/utilized in an electric circuit is known as Power (P), which is measured in watt (W). The Power can be calculated as the product of voltage and current (P = V × I).

1. Introduction

Malaria remains one of the most serious tropical diseases and kills an estimated one to two million people each year worldwide (Snow et al., 2005). Spread of drug resistance has played an important role in the occurrence and severity of epidemic diseases in the world (Majori, 2004). As a consequence of resistance to drugs such as quinine, chloroquine, primaquine and mefloquine, the vehemence of malaria has increased in many endemic regions of the world (Schlitzer, 2007; Basco et al., 1994). One of the biggest challenges to malaria control is the emergence and spread of drug resistant P. falciparum strains. Although human malaria transmitted by female Anopheles mosquitoes has four Plasmodium species as its aetiological agents P. falciparum, P. vivax, P. ovaleand P. malariae, the most widespread and severe disease is caused by P. falciparum, which transiently infects the liver before invading red blood cells of the mammalian host. Clinical manifestations occur at the erythrocytic stage and can include fever, chills, prostration and anaemia, as well as delirium, metabolic acidosis, cerebral malaria and multi-organ system failure, which may be followed by coma and death (Fidock et al., 2004; Deprez-Poulain et al., 2005, De Sa et al., 2009).The success of the natural product-based antimalarials such as quinine and artemisinin has showed the importance of plants as potential source of antimalarial agents. Consequently, numerous efforts to identify antiplasmodial activities from plants traditionally used as remedies for fever and malaria (Chen et al., 1997; Havsteen, 2002;

Introduction

Several hazardous chemicals, microbes, and techniques like recombinant DNA technology are used for research and development work in research Institutes and universities. Therefore, the researchers, technical, helping and supporting staffs as well as the laboratory outputs must be kept free from contaminations. Thus, biosafety is necessary to prevent unintentional exposure/ release of hazardous chemicals, microbes/pathogens, and toxins to the laboratory staff and environment. Following the biosafety guidelines and ethical considerations are essential to maintain safety standards for the researchers as well as the environment. This chapter deals with various biosafety measures and ethical issues to be strictly followed while working in a research laboratory. The required safety level needs to be determined as per the chemical/biological materials are used in the laboratory to keep the researcher/environment safe for sustainable research and developmental activities.

Biosafety

The term biosafety is used to describe the procedure and policies needed to ensure environmental and personal safety. Biosafety refers to the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens, toxins, or their accidental release into the environment. A fundamental objective of any biosafety program is the containment of potentially harmful chemical/biological agents as precautionary safety measures.

Why Biosafety?

With the increasing number of countries adopting molecular biology tools and techniques in life science research and development activities, especially in the areas of agriculture and medicine, the biosafety issues are gaining importance to ensure biological safety for the public and the environment. Conducting research in safe and sustainable manner is not only a personal requirement but essential collective efforts to ensure biological safety for a clean and safe environment. This requires rules, regulations, monitoring bodies, and awareness among the workers as well as the public. Therefore, biosafety per se is an integral part of laboratory research and requires awareness among the researchers so that biological safety can be well taken care from the grassroot level.

Introduction
The advent of antibiotics, beginning with penicillin in 1941, transformed medicine by rendering bacterial infections treatable [1]. However, antimicrobial resistance (AMR) has emerged as a profound threat, enabling bacteria to survive in the presence of drugs designed to inhibit or kill them and these are rooted in ancient microbial ecosystems evidenced by resistome genes in 30,000-year-old permafrost [2]. AMR has been exacerbated by human practices: approximately 50% of antibiotic prescriptions are inappropriate, 70% of global antibiotics fuel agriculture, and environmental pollution creates resistance hotspots [1]. By 2025, AMR directly causes 1.3 million deaths annually, with economic losses projected at $100 trillion by 2050 [1]. Priority pathogens, including carbapenem-resistant Enterobacteriaceae (CRE), methicillin-resistant Staphylococcus aureus (MRSA), and colistin-resistant strains via mcr-1, challenge last-resort therapies, particularly in low-resource settings [3]. This chapter delivers a technically extraordinary exploration of AMR, structured into genetic, biochemical, and evolutionary mechanisms, each elaborated with molecular precision and step-by-step clarity for learners. Genetic alterations provide the DNA-level foundation, biochemical pathways execute molecular defenses, and evolutionary forces drive adaptation and dissemination. It ensures scientific rigor and broad appeal for students, researchers, clinicians, and policymakers.
Historical Context and Global Burden
AMR emerged shortly after antibiotics’ introduction, with penicillin-resistant S. aureus identified by 1942 due to ß-lactamase production [1]. The 1960s

Introduction

The study of infectious diseases, alongside other clinical and natural strengths, is going through fast change introduced on by the approach of reasonable next generation sequencing (NGS) advancements (Koser et al., 2012).These technologies become adopted as routine methods easily, and laboratory techniques are transformed throughout the process. The amount of genomic bacterial data generated is huge. As of this writing, for example, over 200,000 Salmonella genomes alone are in the public domain with hundreds being added weekly. A whole genomic DNA sequence characterizes the maximum practicable level of structural detail on the individuating traits of an microbial organism or population. It can therefore be used to provide more accurate microbial genome recognition, evolutionary relations and a detailed catalogue of epidemiologically important characteristics. This has a significant role to play for outbreak research, diagnosis and treatment of infectious diseases, microbiology and epidemiology (Allard et al., 2019). Besides, DNA sequences are also a universal dataset which can theoretically be used to determine any biological function. This means that antimicrobial resistant (AMR) can be detected and to track the development and spread of AMR bacteria in a hospital or the community.

Introduction
The emergence and spread of antimicrobial resistance has changed the landscape of infectious disease treatment, creating an urgent need for new diagnostic and therapeutic approaches4’7. Traditional phenotypic antimicrobial susceptibility testing, while remaining the gold standard, requires 24-72 hours for results and provides limited insights into resistance mechanisms. Limited turnaround time is often a constraint that requires empirical use of antibiotics, potentially driving the propagation of other resistance emergence as well as suboptimal patient outcomes. Thanks to mass spectrometry, clinical microbiology has greatly benefitted through the rapid detection of pathogens. However, the application is far broader than species identification. The integration of multiple omics fields like proteomics, metabolomics, genomics, and transcriptomics through mass spectrometry provides unprecedented means to unravel, predict, and combat antimicrobial resistance (AMR) on a molecular level. The use of a multiomics approach is demonstrated to show that AMR is a complex phenotype that arises from the intersection between genetic mutations, variations in gene expression, metabolic differences, and changes in protein structures that all act in unison to affect bacterial survival in the presence of antibiotics. The conversion of raw mass spectrometry outputs into clinical insights amenable to decision-making is dependent upon bioinformatics. Through the use of sophisticated computational methodologies, such as machine learning and network analysis, bioinformatics provides the means to incorporate heterogeneous omics datasets. Through this approach, resistance-related biomarkers can be identified, predictions for therapeutic efficacy can be made, and new targets for intervention can be identified. The intersection between high-resolution mass spectrometry, broad-based omic profiling, and strictbioinformatics provides a powerful platform for addressing the intractable challenge of antimicrobial resistance.

The following plants have been worked out in the laboratory of Prof. N. Raaman: Cynodon dactylon, Artemisia pallens, Leptadenia reticulata, Clausena dentata, Tylophora indica, Aristolochia bracteolata, A. indica, Cissus quadrangularis, Hypericum mysorense. and Solanum trilobatum. Out of some of the data of these plants have been included in the previous chapters. In this chapter, compound isolation (CD1) from Clausena dentata (worked by Dr. R. Kamaraj) and characterization of the compound are given in detail.

Introduction Antimicrobial resistance (AMR) has emerged as one of the gravest threats to modern medicine, undermining decades of progress in infectious disease control. It is cited by the World Health Organization (WHO) as one of the top 10 threats to global health. In 2019 alone, AMR was associated with approximately 4.95 million deaths, including 1.27 million directly attributable to resistant bacterial infections. This makes AMR a leading cause of death worldwide, surpassing HIV/AIDS (0.86 million deaths) and malaria (0.64 million deaths) in the same year. Projections suggest that if the trend continues unchecked, AMR could cause 10 million deaths annually by 2050, representing a profound human-cost escalation. Livestock and Animal burden While the quantitative burden of AMR in animals is not yet globally mapped, its effects in livestock are already both profound and alarming. Over 70% of all antimicrobials globally are used in farm animalsfrequently for growth promotion and disease prevention—not just treatment. This widespread use contributes to emerging resistance in key pathogens—Salmonella, E. coli, Campylobacter in poultry, and severe swine dysentery in pigs. In some outbreaks, swine dysentery, resistant to conventional treatment, has caused up to 90% morbidity and 30% mortality in weaned pigs. Animal-to-Human Transmission and the One Health Paradigm Antimicrobial resistance in livestock doesn’t stay on the farm—it seeps into the environment and crosses over to humans via food chains, direct contact, and environmental pathways. For example, substandard veterinary medicines and antibiotic misuse has been traced in zoonotic infections and veterinary-tohuman AMR transmission.Surveillance gaps remain: the World Organisation for Animal Health (WOAH) acknowledges that while human-related AMR mortality is well-documented, the animal health burden is not yet quantified globally—though numerous projects are underway to address this gap. The failure of conventional antibiotics to treat resistant infections highlights the urgent need for alternative therapeutic strategies. Among the most promising options is bacteriophage therapy—the use of viruses that specifically target and kill bacteria. Bacteriophages Bacteriophages, often referred to simply as phages, are viruses that infect and replicate within the bacteria. They are considered as the most abundant biological entities on Earth, with an estimated amount 10³¹ phage particles present in the biosphere—outnumbering bacteria by approximately tenfold. Phages are naturally found in diverse ecosystems including soil, ocean, sewage, and even within the microbiomes of humans and animals.

Phenolic compounds constitute a major class of plant secondary metabolites that are widely distributed in the Plant Kingdom. Plant phenolics are biosynthesized through the shikimate/phenylpropanoid pathway leading to different compound classes. They may occur in their natural sources in free forms, as glycosylated or acylated derivatives, and as oligomeric and polymerized structures, such as hydrolyzable and condensed tannins, phlorotannins or lignins. They may also be found linked to plant matrix components such as cell walls, carbohydrates or proteins. It should be indicated that although the terms “plant phenolics” and “polyphenols” are indistinguishably used by some authors, they are not synonymous.

Toxicology is defined as the study of chemical or physical agents which interact with biologic systems to produce a response in the organisms. Toxicity is the relative ability of a substance to cause injury to biologic tissue. Given the broad range of toxicities any substance might eventually invoke in an organism, it is easy to understand the wisdom of Paracelsus (1493-1541) when he said, “All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy”.

Toxicology is the study of the harmful effects of chemical compounds on biologic systems, including their properties, actions, and effects. The toxic agent is referred to as a toxicant or poison. The term toxin refers to poisons produced by a biologic source (eg, venoms, plant toxins); the redundant term biotoxin is occasionally used. Toxicosis, poisoning, and intoxication are synonymous terms for the disease produced by a toxicant. Toxicity (sometimes incorrectly used instead of poisoning) refers to the amount of a toxicant necessary to produce a detrimental effect. Hazard describes the likelihood of poisoning under conditions of use.

The traditional medicines are actually used in human for number of years and therefore its clinical existence comes as a presumption. However, for bringing more objectivity and also to confirm traditional claims, systematic clinical trials are necessary. In Ayurvedic medicine research, clinical experiences, observations or available data becomes a starting point. In conventional drug research it comes at the last. Thus, the drug discovery based on Ayurveda follows a ‘Reverse Pharmacology’ path546.

Nevertheless, all the critical Pharmacopoeial tests such as dissolution time, microbial, pesticide and heavy metals contamination etc. must be in accordance with global standards. It is important to ensure that all the Ayurvedic medicine manufacture is in accordance with Current Good Manufacturing Procedures for herbal products547,548. There have been concerns about quality standards and safety issues of herbal medicines549. The need for new regulations for botanical medicines has also been frequently stressed and some such regulations are coming in force in different parts of the world550,551.

Learning objectives

1. Recognise inflammatory cell types

2. Recognise common skin pathogens

3. Diagnostic test are best for each case

4. Gain experience in interpretation

5. Recognise important artefacts

″The most crucial step towards healing is having the right diagnosis″. While approaching a case, a clinician should follow appropriate steps in a systematic way for accurate diagnosis of dermatological disorders. A careful history is a first step to choose appropriate diagnostic tests.

ABSTRACT

Purine Nucleoside Phosphorylase (PNP) is an enzyme that functions in the purine salvage pathway by catalyzing the phosphorolysis of purine nucleoside thereby releasing the purine bases and phosphorylated sugar. Interest in PNP as a drug target arises from its role in T-cell proliferation and T-cell mediated autoimmune diseases. Recently, we have isolated the compound, coixol, from the plant Scoparia dulcis L. and its three-dimensional structure has been elucidated. As the structure of the coixol mimics the structure of PNP inhibitors, we have undertaken the molecular modeling studies of this compound with Human PNP, as a screening test for further in vivo studies. Generally PNP inhibitors have been synthesized with a ribose sugar moiety attached to them. As the plant Scoparia dulcis L. contains glucose and rhamnose (six membered ring), these sugar moieties are also included along with ribose in the presen study. In order to understand the binding mode and interactions of the isolated compound attached with different sugar groups, we have carried out the docking studies with Human PNP. The interactions between the ligand molecules and active site residues of Human PNP are similar to those of solved complexes and the energy values are also comparable, which clearly indicate that these ligands may act as inhibitors.

Introduction
Diabetes mellitus is a disease the related to metabolism of the body and malfunctioning of pancreas. Pancreas having alpha and beta cells where alpha cells produce enzymes needed for the digestion and beta cells mainly produce insulin. One important component that regulates blood glucose (sugar) levels and how it is delivered to the body’s cells is Insulin. The body’s cells run out of their primary energy source when insulin levels are low. Insulin voices the body’s cells to engross glucose from the bloodstream. Without an satisfactory amount of insulin glucose cannot invade the cells and so it accrues in the blood, triggering hyperglycemia (high blood sugar). In retort to this ostensible starvation, the body starts flouting down supplies of protein & fat for energy, producing weight loss contempt a ravening appetite. The body efforts to remove the extra glucose by micturating. To avoid thirst, the dog drinks more and more water. Type I increased diabetes mellitus outcomes from completeobliterat ion of the insulin-creating beta cells. In dogs, this is the most prevalent kind of diabetes. As the name implies, insulin injections are necessary to balance blood sugar in dogs with this kind of diabetes.