eChapters

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.

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.

The complement system is a group of about 20 heat labile plasma proteins which constitute nearly 10% of total serum proteins. These are important in activation of phagocytes, control of inflammation and cytolysis. They form one of the major immune defense systems of the body. The complement system plays important role both in innate and adaptive immune system. The main functions of complement are chemotaxis; opsonization and cell activation; immune clearance of immune complexes from circulation; lysis of target cells; triggering and amplification of inflammatory reactions and development of antibody responses. Evolutionary, the system is very ancient and is present even in invertebrate organisms like starfish and worms.

Major Histocompatibility Complex (MHC) is a closely linked cluster of genes which have codes for cell surface proteins known as  MHC molecules. These molecules have very important role in presentation of processed antigens to the T cells in initiation of an immune response as T cells recognize an antigen only when it is presented along with MHC molecule. These are highly polymorphic and are the major barrier to organ/tissue transplantation. All vertebrates have MHC. MHC differs among members of a species and are thus called alloantigens. MHC participates in the development of both humoral and cell-mediated immune responses.

Microbiology is the branch of science which deals with the diverse minute forms of life such as bacteria, archaea, viruses, fungi, prions, protozoa and algae. The study includes structure, classification, growth and nutrition, natural habitat, genome, antigenic characteristics, virulence determinants, types of toxins produced, pathogenesis, diagnosis and ways to combat the infection caused by the microbes.

The genetics is the study of inheritance and variability of organisms. Bacterial genetics is a sub-field which specifically studies transmission of genetic information from one bacterium to its progeny or between the interbreeding bacteria and the expression of genetic information.

Mutation

Hugo de Vries (1848-1935, Dutch botanist) coined the term ‘mutation’. It is originated from a Latin word which means ‘to change’. The heritable change in bacterial DNA sequences is known as bacterial mutation. The mutation will generate a new type of bacteria with altered genotypical and phenotypical characteristics, known as ‘mutant’. The parent bacterial cell from which the mutants are generated is known as ‘wild type’. In nature bacterial mutation is a rare incidence. The spontaneous mutation occurs at a rate of 1 in 10⁵ to 10⁸. The phenotypical expression of mutation occurs quickly as the bacteria are haploid with shorter generation time.

O    :    Pint.

O antigen      :    A polysaccharide antigen extending from the outer membrane of some gram-negative bacterial cell walls; it is part of the lipopolysaccharide.

O&E    :    Observation and examination.

Oa    :    Open access.

In 1850, Bacillus anthracis was first reported by three individuals in two different countries i.e. Delafond and Davaine in France, and Pollender in Germany. They could not establish the relationship of the bacterium with the infection. Absolute proof of the anthrax etiology as B. anthracis was established by Robert Koch during 1876-77. The name Bacillus anthracis was first coined by Cohn (1875) and the disease ‘anthrax’ was first named by Hippocrates (5th century B.C.). In Greek language meaning of ‘anthrax’ is ‘coal’ referring to the black coloured skin or blood in affected animals.

Antony Van Leeuwenhoek (1681) first observed the spirochetes under the microscope and described them. In 1833, Ehrenberg used the name Spirochaeta for free living spiral shaped organisms. Obermeir (1873) first described a spirochete of pathogenic significance isolated from human patients with relapsing fever. Inada (1916) first isolated Leptospira icterohaemorrhagiae from human patients of Weil’s disease. In 1980, Nogochi proposed the name as Leptospira.

In India, Taylor and Goyale (1931) first reported Leptospira from human patients suffering with jaundice in Andaman Islands. Ayyar (1932) first reported Leptospira from dogs in Chennai city.

Feeds and feeding of dairy animals are arguably the most important part of the dairy farm management. And it is not only what we feed that is important but how we manage our feeding is equally as critical. What we feed today not only determines the animal’s milk production but also influences their entire lactation cycle that follows. Dairy animal feeding with balanced ration influences health status, reproductive, productive performance and better economic return in organic environment, all of which are critical to a profitable and sustainable dairy business as well as welfare of the dairy farmers. Feed costs currently account for around 45 percent of total milk price on average, with concentrates representing around 35 percent and forages around 10 percent.

Identification of enteric bacilli is of prime importance for controlling intestinal infections. This can be achieved by preventing contamination of food and water by a group of bacteria belonging to family – Enterobacteriaceae which are found in the intestinal tract of human beings and lower mammals. The microorganisms of this family are short, gram negative, non spore forming bacilli. Such common enteric bacteria are a) pathogens such as species of Salmonella and Shigella b) occasional pathogens like species of Proteus and Klebsiella and c) normal intestinal flora like species of Escherichia and Enterobactor.

The increasing use of antibiotics in livestock and poultr y led to development of antibiotic resistant micro organisms and antibiotic residues in livestock products besides other side effects. These caused alarm bells ringing all over the world and by the end of June, 1999 majority of antibiotics as growth promoters in monogastric diets had been banned within the EU. The ban on use of antibiotic at sub therapeutic level in Europe and the potential for a ban in the United States have made scientists to seriously think of alternatives to antibiotics. In fact, the use of probiotics for farm animals was already stimulated by the findings of the Schwann Committee in 1969 which recommended that antibiotics in animal feeds should be restricted. However, the concept of probiotics gained momentum in later part of the last century. In recent years, with the public disapproval of using antibiotics in livestock and poultry production due to their residual effects in their products, there has been a greater emphasis on the use of probiotics in poultry.

A. Answer the following.

Q.1.     What is the percentage of different subclasses of IgG in the serum ?
Ans.     IgG1 : 65 ; IgG2 : 24 ; IgG3 : 7 ; IgG4 : 4

Q.2.     How many inter heavy chain bonds are there in different subclasses of IgG ?
Ans.     IgG1 : 2 ; IgG2 : 4 ; IgG3 : 13 : IgG4 : 2.

Q.3.     What is the half life of different subclasses of IgG ?
Ans.     IgG1, IgG2 and IgG4 : 23 days ; IgG3: 8 days.

Innate immunity or innate immune response is a set of non-specific or less specific mechanisms which provides the first and rapid line of defense. All such mechanisms exist prior to the exposure to a pathogen i.e. before the onset of the infection. It is also called inborn or innate immunity. The different components of the innate immunity like physical barriers, chemical barriers, phagocytosis, inflammation etc. act to prevent the entry or elimination of the microorganisms in a non- specific manner.

Humoral immune response is the immune response which is mediated by antibodies.. It is called humoral immune response because antibodies are transferred with the ‘humour’ that is fluid or blood. Antibodies are the proteins produced by B lymphocytes.

The chief functions of Clinical Microbiological laboratory are to examine and culture specimens for microorganisms, to make accurate species identification of important isolates and to perform antibiotic susceptibility tests when indicated. These tasks will assist physicians in the diagnosis and treatment of infectious diseases.

Once the specimen is received in the laboratory, the specimens are examined visually depending on the physicians order and the nature of the specimen. Wet mounts and smears may be prepared and stained for microscopic examination. Observations may or may not be immediately reported to the physicians depending on the definitiveness of the results. Timely information may often be used to establish a presumptive diagnosis and institute a specific course of therapy. Specimens that require definitive identification of potentially pathogenic microbes are processed further. All agar plates are streaked for colony isolation; then plates and broths. All cultures are placed in an incubator with appropriate temperature and environmental conditions to maximize the growth and replication of microbes. Often a presumptive microbial identificationcan be made. A final report should be delayed while subcultures and additional test procedures are performed to identify the organisms definitively.

Preliminary Identification of Unknown Bacteria
A number of techniques may be used in the direct microscopic examination of clinical specimens to demonstrate the presence of microbes The examination of wet mounts of unstained materials by phase contrast or
dark field microscopy is useful for demonstrating motility, spirochaetes and endospores. Giemsa, Wright or acridine orange stains may be helpful in observing bacterial forms that stain poorly or that have little contrast from
back ground material.

The complement system is a group of about 30 heat labile plasma proteins which constitute nearly 15% of total serum proteins. It is a system of plasma and membrane-associated serum proteins which can elicit a highly efficient and regulated inflammatory and cytolytic immune responses to different types of infectious organisms.