eChapters

There are other groups that contribute to the reactivity of enzymes beside amino acid residues.

These groups are called cofactors - chemicals required by apoenzymes (inactive) to become holoenzymes (active).

There are two types of cofactors:

1. Essential ions - metal ions -inorganic
2. Coenzymes - organic molecules that act as group-transfer reagents (accept or donate groups)- can also be H⁺ and/or e^-

Both provide reactive groups not found on amino acids side chains.

Coenzymes can be either cosubstrates (loosely bound to enzyme; is altered, then regenerated) or prosthetic groups (tightly bound to enzyme).

Coenzymes can be classified by their source:

HELMINTHS Collection of parasites Fresh helminth parasites should be collected, as far as possible to achieve better results in staining. This can best be done by conducting a post-mortem examination of the animal for which the procedure given below should be followed. On opening the body and before removal of the viscera, examine the body cavity (for free lying parasites, e.g. Setaria), subcutaneous tissue (for Parafilaria and Hypoderma larvae) and the surfaces of internal organs for spot or nodules that might contain parasites. Remove them for histopathological examination (in 10 % formalin). Remove the visceral organs separately and place them in different enamel trays.

On the face of an outbreak of a disease, collection of right clinical specimens in proper preservative, their transportation to the laboratory under ideal conditions and quick processing of specimens in the laboratory to identify the causative agent are crucial to successful control of the disease. This attains more importance when a highly contagious disease appears.

Collection of specimens from a virus-infected host is the first step for demonstration or isolation of a virus. Specimens for virus isolation should be collected at the time when the maximum amount of virus can be expected to be present. i.e. collect the specimens as early as possible after the onset of clinical symptoms. In many viral diseases maximum viral production occurs before the patient becomes ill so that it is ideal to collect the specimen at an even early time.
The nature of the specimen taken in any particular instance is determined by the patient’s clinical syndrome and previous history. They are best considered under three main headings.

1. Specimen for virus isolation/antigen detection/ nucleic acid detection
2. Specimen for serological investigation
3. Specimen for direct examination

To identify parasitic infections, it is essential to examine excretory- secretory products (ESPs), viz., faeces, urine, sputum and nasal discharges as examination of such material is essential for clinical diagnosis. It is extremely rare to get an animal completely free from parasites and at the same time all animals may not show signs of disease. Most of the important helminths and coccidia inhabit intestinal tract. Therefore, clinical diagnosis of gut dwelling helminths and coccidian depends mostly on identification of ova/oocyst in faecal samples

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.

A. Answer the following.

Q.1.     Why the serum is inactivated at 56º C for 30 min before using it in CFT and VNT ?
Ans.     The complement and non-specific virus inhibitors are inactivated in this process.

Q.2.     What are the different enzyme and complement factors are secreted continuously by macrophages ?
Ans.     Lysozyme and Complement 2, 3, 4 and 5.

B. Write true or false about the followings.

Q.1.     The classical complement activation pathway is dependent on antigen-antibody complex.
Ans.    True.

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.

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.

This chapter introduces the complement system as an important component of innate and adaptive immunity. It explains the classical, alternative and lectin pathways of complement activation and identifies the molecules involved in each pathway. The formation and functions of C3 convertase, C5 convertase and the membrane attack complex are discussed. Questions address complement-mediated cell lysis, inflammation, chemotaxis and opsonization. The roles of C3b, C3a, C4a, C5a, properdin, factor H and other regulatory proteins are also examined. By covering the activation and regulation of complement, the chapter explains how this system enhances antibody activity and helps eliminate invading microorganisms

Introduction

Until the 20thcentury, bacterial infections were the number one cause of human death. In the 1940s, antibiotics became the lifelines for treating serious infections that saved millions of lives. Despite the advantages derived from the use of antibiotics, resistance to these drugs emerged. Unfortunately, resistance to most antibiotics emerged in time-course even for the most powerful antibiotics developed.Resistance to antibiotics is a challenge to the scientific community in keeping up the pace of antibiotic discovery.World Economic Forum’s Global Risks Reporthas listed antibiotic resistance as one of the greatest threats (WEF, 2020).United Nations (UN) has warned that drug-resistant diseases could account for 10 million deaths each year by 2050. By 2030, antimicrobial resistance (AMR) could likelylead up to 24 million people into extreme poverty(IACG,2019). Currently, at least 7,00,000 people die each year due to drug-resistant diseases (IACG,2019). The emergence and widespread of antibiotic-resistant organisms is a global challenge and needs a multi-dimensional approach to address the fundamental issues to minimize drug-resistance.The Antibiotic Resistance Genes Database (ARDB) contains a repository of around 23,000 antibiotic resistance gene or protein sequences conferring resistance against over 240 antibiotics (ARDB). Computational Biology (Bioinformatics) has become one of the important tools in studying antimicrobial resistance. It employs a range of computation techniques to make predictions about the biological phenomenon.

For looking into the biosafety in research laboratories, agriculture and other food producing industries it is important to look into the numerous strains of lactic acid bacteria that are associated with food systems and are capable of producing bacteriocin or antibacterial proteins with activity against foodborne pathogens such as Listeria monocytogens, Staphylococcus aureus and Clostridium botulinum. Recently considerable attention has been given on the identification of these proteins and their biochemical properties including spectrum of activity, production conditions, purification procedures, aminoacid composition and mode of action. Advances in genetic engineering have facilitated the characterization of proteins at genetic level, providing information about protein synthesis, structure and cloning of bacteriocin genes into other organism. One problem in the use of bacteriocin in their applications as bio-preservatives is the emergence of resistant population. The understanding on the mechanism of resistance to bacteriocins is limited which limits the application in biopreservation. Much of the work on bacteriocin has been done under controlled laboratory conditions without the presence of adventitious microbial population. More research is needed to determine the efficacy of bacteriocin producing organisms and bacteriocins which are stable under commercial conditions. Foodborne illness is a major international problem and an important cause of reduced economic growth. The contamination of the food supply with the pathogens and its persistence, growth, multiplication and/or toxin production

Various physical, chemical and biological factors interdependently affect the fungal colonization or production of the mycotoxin. Physical factors such as temperature, relative humidity and insect infestations create ideal environment for fungal colonization and mycotoxin production. Stresses such as drought, an increase in temperature, and an increase in relative humidity may selectively alter colonization and metabolism of mycotoxigenic fungi and thus alter mycotoxin production (Russell et al., 1991). Cool and damp weather favours the germination of the sclerotia and thus ergot alkaloid formation in fescue and ryegrass.

Biological factors are categorized into intrinsic factor such as fungal species, strain specificity, strain variation and instability of toxigenic properties. Interaction between the toxigenic fungal species and substrate affect the fungal colonization and mycotoxin production. Some plant species are more susceptible to colonization and some resistant plant species may also get infestation due to favourable environmental conditions.

Review of literature or consulting of literature is just beginning of a research project. The main intellectual work is to study the previous work meticulously and to draw conclusions regarding existing problems. This leads us to another intelligent part of the research i.e. planning of the research work. If you analyze what you are planning, it is only about materials to be employed and methodology of the research work (with planning of the work)- only these three parameters are under your control as results will be the outcome of these three facets while discussion will be how you consider your results in presence of previous research work. In fact, this statement of mine is more align to the sermon of “Shrimad Bhagwat Geeta” (5) where it is stated that man has control only on his actions (planning and conducting research) hence fulfill this duty without caring for the results-which in turn will depend on your analyzing the previous facts and how you have planned to solve the problem. Therefore, pay full attention on these three facts of the research.

The economic loss in livestock arising due to mycotoxicosis is enormous apart from being a health risk. Indian economy is heavily affected by mycotoxins. It was estimated that 10 million dollars were lost in India’s export within a decade due to groundnut contamination with mycotoxins (Vasanthi and Bhat, 1998). Many developing countries have realized that reducing mycotoxin contamination in foods will not only improve the health and production in animal, but also increase the raw material exports (international trade) to other countries. The following strategies are to be adhered strictly to reduce the mycotoxins contamination of crops in the field.

1. Early harvesting

Early harvesting reduces the fungal infection of crop in the field. Early harvesting and threshing of groundnuts results in lower aflatoxin levels and higher gross returns of 27% than in delayed harvesting (Rachaputi et al., 2002).

Sterilization refers the complete removal or elimination of microorganisms from an environment. Physical and chemical agents are available to remove the microorganisms from the environment. Heat, irradiation and filtration are the three physical agents and various chemicals are available for sterilization.

Terms related to control of microorganisms

Cide or cidal means to kill the microorganism (Ex. Bactericidal refers the killing of bacteria).

Static or stasis means inactivation or inhibition, but not killing. They prevent multiplication of the organism. (Ex. Fungistatic refers the inhibition of growth of fungi.).

Sepsis refers break down of a living tissue by organisms and is accompanied by inflammation and pus formation.

Antispetic is an agent applied externally on living tissues to kill or inhibit the growth of the organisms.

Disinfectant is an agent applied externally on inanimate objects (not living tissues) to destroy harmful pathogens in their vegetative stage.

(Antiseptics are milder than disinfectants because to avoid the side effects)

Sanitation refers removal of organisms from a location by cleaning but not by sterilization.

Introduction
Antimicrobial resistance (AMR) poses an escalating threat to global health, eroding the efficacy of antibiotics and complicating the management of bacterial infections. The proliferation of resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum betalactamase (ESBL)-producing Enterobacteriaceae, and multidrug-resistant Mycobacterium tuberculosis, has driven significant increases in morbidity, mortality, and healthcare costs. The ability to detect AMR accurately and promptly is paramount for guiding clinicians in selecting effective therapies, implementing targeted infection control measures, and informing public health strategies to curb the spread of resistant organisms. Conventional methods, rooted in phenotypic assessments, have served as the cornerstone of AMR detection in clinical microbiology laboratories for decades. These techniques evaluate bacterial growth under controlled exposure to antibiotics, offering direct evidence of resistance or susceptibility that molecular approaches, which focus on genetic markers, may miss due to silent mutations, complex regulatory mechanisms, or novel resistance pathways. This chapter provides an exhaustive examination of conventional AMR detection methods, encompassing disk diffusion, broth and agar dilution, gradient diffusion, and automated susceptibility testing systems. These approaches, refined through decades of clinical and research application, remain indispensable for their reliability, cost-effectiveness, and adaptability across diverse settings, from well-resourced tertiary hospitals to laboratories in low-income regions. By exploring their principles, procedures, applications, and limitations, we aim to underscore their enduring significance amidst the rise of rapid molecular diagnostics. The discussion also addresses specialized tests for detecting specific resistance mechanisms, such as beta-lactamase production, inducible clindamycin resistance, and carbapenem’s activity, which are critical for tailoring antimicrobial therapy. Through a critical lens, this chapter evaluates the strengths and challenges of these methods, offering insights into their role in addressing the global AMR crisis, their integration with emerging technologies, and their continued relevance in both routine diagnostics and epidemiological surveillance. The value of conventional methods lies in their ability to translate laboratory findings into actionable clinical decisions. Unlike genotypic methods, which detect resistance genes but may not predict phenotypic behaviour, these techniques assess how bacteria respond to antibiotics under conditions mimicking in vivo environments, aligning closely with therapeutic outcomes. As resistance patterns grow increasingly complex, driven by mechanisms like carbapenem’s and efflux pumps, conventional methods provide a practical foundation for diagnostics, particularly in resource-limited settings where advanced molecular tools are often inaccessible. Their role in global AMR surveillance, such as through the World Health Organization’s Global Antimicrobial Resistance Surveillance System (GLASS), further underscores their importance in tracking resistance trends and informing policy.

Introduction
Q fever is a zoonotic bacterial infection caused by Coxiella burnetii, an obligate intracellular parasite, classified within the family Rickettsiaccae. Q fever (Q for “query”) was first used in 1937 to describe a mysterious febrile illness of packing house workers in Brisbane, Australia. The causative agent was isolated from infected workers and later was identified as Rickettsiae. Almost simultaneously the same organism was identified wood tick collected in Montana. Cox, an American, and Burnet, an Australian, where honored for their early work with this organism, hence the name “Coxiella burnetii”.
Host and Susceptibility
Coxiella burnetii is a well-established infectious agent that has reached a state of balanced pathogenicity in a plethora of host. Human are unnatural and usually dead-end-host. The microorganism generally maintained in a less pathogenic form in separate cycle existing independently among wild mammals and their haematophagus arthropods, principally tick and in domestic animals.
Morphology
It is an intracellular organism which is a polymorphic bacillus (0.2-0.4mm width, 0.4-1.0 mm length), which has a cell membrane, like Gram negative bacteria. However, it stains poorly with pigmented Gram stain, but gimenez staining is traditionally used to stain the Coxiella burnetii pathogen from pathological materials and crop. Coxiella burnetii has several distinctive characteristics, including a sporulationlike
process that protects the organism against the external environment, where it can survive for long periods. In mammals, the usual host cell of C. burnetii is the macrophage, which is unable to kill the bacterium. The other an important characteristic of C. burnetii is its antigenic variations, those called phase variation. This antigenic shift can be measured and is valuable for differentiating acute from chronic Q fever. Thus, it displays two antigenic phases those are phase-I and phase- II that are liable to the Lipopolysaccharide (LPS) of the membrane.

14.1 Culture from Urine

Infection of urinary tract due to microorganisms affect patient of all age group and both sex., and they vary in severity from an unsuspected infection to a condition of severe systemic disease. Urine is a ordinarily excellent culture medium for the multiplication of common pathogen of urinary tract. Most bacteria found in the urine are due to contamination of skin flora during passage and they tend to increase greatly, exceeding 10⁶/ml. When the bacterial count exceeds 10⁵ per ml of urine it indicates significant bacteriuria and is indicative of a urinary tract infection. Infection of the urinary tract may be limited to a single tissue/ organ or they may spread upward and involve the entire urinary system. Escherichia coli is the most common urinary tract pathogen. Pseudomonas aeroginosa causes another important urinary tract infection, which is characterized by blue green pus. Cystitis (inflammation of urinary bladder), Pyelonephritis (inflammation of the kidney) are permanent diseases of urinary tract.

Urinary tract infections are generally characterized by bacteriuria, bacteria in the urine. Quantitative microbiological assay of urine can identify the probable pathogen. For this purpose, calibrated loop- direct streak method and pour plate method are routinely used. Now-a-days commercially prepared special plastic device called the urine dip slide and diagnostic urine–culture called Bacturcult is being used for isolation and identification of microorganisms from genitourinary tract infections. The Bacturcult is a sterile disposable plastic tube coated with a special medium on its inner side for detection and identification of bacteriuria.

Introduction 
The discovery and introduction of antimicrobial agents to clinical medicine was one of the greatest medical triumphs of the 20th century that revolutionized the treatment of bacterial infections. However, the gradual emergence of populations of antimicrobial resistant pathogenic bacteria, resulting from use, misuse and abuse of antimicrobials has today become a major global health concern. Antibacterial drugs have revolutionized our ability to control bacterial disease, and their clinical availability has led to dramatic decreases in morbidity and mortality. (Walsh and Wright, 2005). As such, these therapeutics underpin modern medicine. Despite the integral role of antibiotics in sustaining our modern lifestyle, they are undervalued in both cost and significance by society. Over the past century, their use has provided strong selective pressure on microorganisms, leading to preferential survival and spread of those harbouring antibiotic resistance mechanisms. Multidrug resistance is now commonplace amongst bacterial pathogens with antibiotic resistance now affecting all antibiotic classes. (Coates et al, 2011). This is particularly worrisome in the case of Gram-negative bacteria (e.g. Pseudomonas aeruginosa and Acinetobacter baumannii) for which treatment options are already limited. The ‘broken’ economics of antibacterial research and development is often quoted as the main reason for the lack of new therapies but the truth is that it is hard to discover new antibacterial drugs, and the science is not sufficiently advanced to allow discovery of efficient and effective drugs. This has led to fears of a ‘post-antibiotic era’ as it has been estimated that 5–20 novel antibacterial drugs need to enter clinical development in order to effectively contend with the current resistance problem. However, given the attrition rate within the existing drug discovery model, at least 200 discovery programmes would be needed in order to achieve this outcome. Hence, new approaches to antibiotic discovery are needed. The antibiotic pipeline The antibiotic pipeline is not what it once was.4 Pharmaceutical companies were once the main provider of novel antibiotic molecules but they largely withdrew in the late 1990s because of the lack of success and low financial returns in bringing new antibacterial drugs to the market.5 The environment of discovering and developing new antibiotics was quite different during the so-called ‘golden era’ of drug discovery. Antibiotics worked remarkably well because resistance was low and physicians had access to a variety of efficacious antibiotics. Antibiotic research and development programmes were inclined to focus on improved pharmacology to achieve less frequent dosing e.g. once a day, rather than innovative new antibiotics. Natural product screening strategies tended to result in rediscovery of compounds rather than finding new ones. There was also no need to consider those natural products with undesirable properties such as toxicity. Today, only a few large companies such as GlaxoSmithKline, Novartis, Merck and Roche are still actively engaged in antibiotic R&D, with many of the original antibiotic provide.The natural world remains the largest source of novel drug scaffolds making this a viable option in the search for new antibiotic compounds. Advances in bacterial culture techniques, molecular biology and metagenomics will make natural product drug discovery easier and more costeffective, obviating these limiting factors. Screening procedures must include whole-bacterial cell assays, addressing the issue of bacterial permeability and efflux early in the discovery process (Shore and Coukell, 2016). Additionally, the generation of training schemes by, and with, pharmaceutical companies that cover all aspects of the pipeline and include natural product drug discovery, are essential and will ensure that expertise is passed on to future researchers. Investment should also be made into the study of previously characterized lead compounds that did not reach the clinic, so called old leads. The reasons that led to these compounds being dropped from further development vary, ranging from financial issues to trial design, dosing problems and toxicity. It may be that there is now sufficiently improved technology and expertise to develop these as safe and efficacious antibacterials. The revival of interest in old leads could also provide an additional source of novel antimicrobials. A freely accessible database of antibiotics that were never developed has recently been launched, Antibiotic with the aim to reduce unnecessary duplication of Pharmaceutical agents such as nanospheres, liposomes, and immunoglobulins for example have been developed which can alter the pharmacodynamics of a compound, act as carriers for sustained or controlled drug release, and for immunization therapy.