eChapters

Injudicious and indiscriminate use of several agrochemicals viz. pesticides, herbicides, insecticides, dyes and medicaments in the field of agriculture and veterinary practices threatens the human and animal population for the toxicity of these environmental deterrents. These agrochemicals have been associated with several episodes of accidental poisoning and mortality among human and animal population. The episode of Mass ethion poisoning in Gujarat is one of the commonest example of inclusion of pesticides or agrochemicals in the environmental pollutants. There are modern tools to evaluate and analyze these chemicals in the environmental pollutants of human and animal use. The sensitivity of these modern highly sophisticated tools has now immensely changed the analytical environmental pollutants industry. Gas chromatography and Mass Spectrophotometry i.e. (GC-MS) technique is employed to detect the volatile residues from environmental pollutants. Whereas, High performance liquid chromatography i.e. HPLC is employed to analyse and detect non volatile drug residues. Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical mixtures (the GC component) and identifies the components at a molecular level (the MS component). It is one of the most accurate tools for analyzing environmental samples.

Mesenchymal Stem Cells

Recent advances in the field of regenerative medicine have opened up newer therapeutic modalities using cell-based and cell-free therapies, particularly stem cells, as potential candidates. As per the definition, stem cells are the undifferentiated progenitor cells that can regenerate and can also differentiate into various specialized cells (Hoang et al., 2022). They can be classified as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and postnatal adult stem cells (Locke et al., 2009). Among postnatal adult stem cells, mesenchymal stem cells (MSCs) have emerged as a popular choice of candidate for therapeutics due to the relatively easy availability, simple isolation techniques, low risk of teratogenicity, minimal ethical and legal concerns unlike embryonic stem cells, hence, are easy to apply in clinical research and practice (Voga et al., 2020). Among MSCs of different origin, bone marrow and particularly adipose tissue-derived stem cells (ADMSCs) received more attention and are one of the most common and promising stem cell types. These cells can be simply obtained from subcutaneous and omental fat or liposuction aspirates for the treatment of diverse clinical applications (Kim &Heo, 2014). These cells can also be expanded vigorously in vitro and differentiated into specific cell lineages such as adipocytes, osteoblasts, chondrocytes, and neurocytes and are even genetically stable in long-term cultures (Miana& González, 2018). Mesenchymal stem cells (MSCs) are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the (ISCT) International Society for Cell and Gene Therapy (Dominici et al., 2006).

Introduction

Acute lung injury (ALI) is the manifestation of an inflammatory response of lung resulting from severe sepsis, bacterial or viral pneumonia, trauma, and burns in animals (Ali et al., 2020).It is distinguwashed by disruption to the alveolar capillary membranes, atelectasis of the pulmonary airspaces, and neutrophil and protein-rich fluid infiltration into the alveolar space (Li et al., 2019). Although advances in treatment, the disability and fatality rates of ALI remain high, that is around 30-40% (Ali et al., 2020). At the moment, new therapeutic alternatives are urgently required to treat ALI. As a result, this is a very active area of research to understand the molecular principles underlying pulmonary inflammation and ALI (Reiss et al., 2012). The use of animal models of inflammatory lung has allowed for the probing of pathways of inflammation responsible for injury. Specially, rodent models may serve a link between in vitro research to large animals and clinical studies by allowing for high quantities of samples under physiological circumstances at low expenses (Domscheit et al., 2020).LPS, found in the outer membrane of gram-negative bacteria, consists of a polar lipid head group (lipid A) and a chain of repeating disaccharides (Gao et al., 2023). LPS induced lung injury model, closely mimic sepsis and inflammatory lung diseases, is a well-establwashed model in this field of research (Reiss et al., 2012). To mimic acute lung injury, LPS can be administered either directly through intratracheal or intranasal route (direct pulmonary insult) or indirectly through intraperitoneally and intravenous routes (indirect extra pulmonary insult) (Perl et al., 2011). This chapter focuses mostly on rodent model of acute lung injury caused by lipopolysaccharide (LPS), both direct and indirect. Additionally, it also includes the characteristics and underlying molecular mechanism of LPS induced lung injury along with applications in drug development.

Spontaneous contractions of the uterus are controlled and coordinated for various reproductive functions. The non-pregnant uterus facilitates ova and sperm transport, provides adequate embryo placement for implantation, and contributes to the expulsion of menstrual debris through increased contractility, or periods of relative quiescence (Hutchings et al., 2009). Uterine dysmotility has been implicated in the pathogenesis of certain infertilities, implantation failure, endometriosis, dysmenorrhea, and abnormal menstrual events (Kunz and Leyendecker, 2002; Aguilar and Mitchell, 2010). Although these conditions may not contribute to mortality but can significantly affect patient’s reproductive health, and considerably alter their quality of life (Jones et al., 2004; Weissman et al., 2004; Chachamovich et al., 2010). Dysmenorrhoea refers to the occurrence of painful cramps in the lower abdominal region during menstruation and is one of the unresolved gynecological disorders in adolescent girls and women of reproductive age (Wang et al., 2004; Harel, 2008). This condition is mainly characterized by cramping pain in the lower abdomen immediately before or during menstruation, which does affect the quality of their life and work (Davis and Westhoff, 2001).

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows:

1. Preparation of primary cells culture as described in earlier methods.

2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method.

3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent.

4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.

Genotoxicity potential of any environmental pollutants can be investigated using the micronucleus (MN) test. It is among the most sensitive DNA damage indicators and it has been applied to several organisms and tissues for evaluation of environmental contaminants (Lemos et al., 2011). Micronuclei (MN) assay is more rapid and simpler than chromosomal analysis (Gandhi et al., 2003). Compared to the chromosome aberrations, the final result is relatively easily scored and thus requires less time to make an assessment of the clastogenicity of a chemical (Garriott et al. 2002). An in vitro MN assay can detect the clastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction (Corvi et al., 2008). Although, micronuclei constitute well-characterized biomarkers of chromosomal damage (Salazar et al. 2009) but in vitro MN test does not provide information on the origin of micronuclei (Corvi et al., 2008).

Experimental Procedure

Micronuclei assay is carried out in PMNC and splenocytes as per method of Hayashi et al. (1983).

The concept of single cell micro-gel electrophoresis (SCGE)/comet assay is f irst introduced by Ostling and Johanson (1984) as a method to measure DNA single-strand breaks under neutral conditions. Singh et al. (1988) modified this technique by using alkaline conditions (>13 pH) of electrophoresis, which enabled detection of not only frank strands breaks but also alkali labile sites, DNA cross- linking and incomplete excision repair sites. Now-a-days, this technique is extensively used to study the genotoxic effects of various chemicals. The advantages of SCGE technique include: (1) collection of data at the level of individual cell, allowing for more robust types of statistical analysis; (2) need for small number of cells per sample (<10,000); (3) its sensitivity for detecting DNA damage; and (4) that virtually any eukaryotic cell population is amenable to analysis. The idea is to combine DNA gel electrophoresis with fluorescence microscopy to visualize migration of DNA strands from individual agarose-embedded cells. If the negatively charged DNA contained breaks, DNA supercoils are relaxed and broken ends are able to migrate toward the anode during a brief electrophoresis. If the DNA is undamaged, the lack of free ends and large size of the fragments prevented migration. Determination of the relative amount of DNA that migrated provided a simple way to measure the number of DNA breaks in an individual cell.

Genotoxic effects of any xenobiotics in PMNC and splenocytes of Wistar rats can be studied according to the working protocol of Dhawan et al.

Apoptosis is triggered by extrinsic and intrinsic stimuli as radiation, oxidative stress and genotoxic chemicals. It is defined by characteristic changes in the nucleus morphology including chromatin condensation and fragmentation, overall cell shrinkage/rounding and formation of apoptotic cell bodies. Among the many markers of apoptosis, DNA fragmentation is considered a hallmark. The DNA ladder assay described here is a simple, sensitive, cost-effective and rapid method for estimating apoptosis in cells.

Isolation of Genomic DNA

Genomic DNA from primary cells culture of PMNC and splenocytes of rats is isolated by using standard phenol-chloroform DNA isolation protocol of Sambrook and Russel (2001) as described below:

1. Preparation of primary cells culture of different cells, namely-PBMNCs, thymocytes and splenocytes form Wistar rats and viability checked by trypan blue dye exclusion test.

2. 106 cells/ml of primary cell culture is used and seeded in 24 well culture plates containing along with treatment with xenobiotics at different µM concentration.

3. Cell culture plates are incubated in CO2 incubator at 37°C with 5% CO2 for 12 hrs.

4. The cells are collected in 1.5 ml of micro centrifuge tube and centrifuged at 3200 rpm for 10 min at 4°C; supernatant is discarded and pellet is washed with PBS (pH 7.2).

5. DNA extraction buffer (500 µl/tube) is added in tubes. Gently tap to disperse the cell pellet in extraction buffer and keep in water bath for 1.0 hr at 37 °C (it is to ensure that pellet is completely suspended so that cells are accessible to SDS and Proteinase K).

6. 10 % SDS is added (20 µl/ml) to cell suspension and the tubes are gently mixed by inverting the tube. The contents of tube become viscous which indicates lysis of cells. After that sample is handled gently from onward to avoid shearing of DNA.

7. Proteinase K (15 µl) of 20 mg Proteinase K/ml of buffer is added in tubes in two pulse i.e. half the requirement is added to tube in 1st pulse, mixed gently end to end and kept in water bath at 50°C. After 3-4 hrs, the second pulse of the remaining amount of Proteinase K is added and tubes are incubated at 50°C overnight.

8. Equal volume of equilibrated phenol (Tris saturated phenol pH> 7.8 ) is added in the tubes next day morning. Mix the contents of tube gently by inverting the tube for 15 min till a light coffee coloured uniform solution without any balls of phenol is formed and then centrifuge at 3400 rpm for 15 min.

Safety is a prime concern for humans and animals. Without evaluating the safety of a chemical, no regulatory authority in the world allows the chemical to be used by humans. There are stipulated guidelines for toxicity evaluations. Among the toxicity assays genotoxicity assays are important and have occupied centre stage due to increase in understanding of the genome.

Genotoxicity describes the property of chemical agents to damage the genetic information within a cell to cause mutations, which may even lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic; however, not all genotoxic substances are mutagenic. Generally and depending on the agent, a cytotoxic agent may be genotoxic (causing DNA damage), mutagenic (causing gene mutation) or it may be harmful for cell organelles (e.g. cell membrane) or it may even be teratogenic. Therefore, every cytotoxic agent is not genotoxic.

Why we Need to Evaluate Genotoxicity?

Toxicity can be at any level according to the nature of chemicals and type of exposure. Duration of toxicity can also be short lived or long lived within a generation or continue to next generation. Genotoxicity effects are able to continue in next generation(s), in which the exposed population may not suffer seriously but the next generation may suffer without being exposed to the same chemicals. Therefore, genotoxicity evaluations need serious attention.

Environmental agents (xenobiotics) that are injurious to cells trigger a spectrum of responses that can range from cell death to adaptation, repair, and proliferation. Cell death, in turn, can take the form of apoptosis (a programmed form of cell death). Xenobiotics have been implicated in toxic cell injury, among them free radicals (nitrogen, oxygen, and carbon derivatives) are known to interact with cellular constituents including membrane lipids and chromagen materials.

Cytotoxicity

Cytotoxic: Toxic to cells, cell-toxic, cell-killing. Any agent or process that kills cells. The prefix cyto- denotes a cell. It comes from the Greek “kytos” meaning hollow, as a cell or container. Toxic is from the Greek “toxikon” = arrow poison.

In nutshell, cell cytotoxicity refers to the ability of certain chemicals or mediator cells to destroy living cells. By using a cytotoxic compound, healthy living cells can either be induced to undergo necrosis (accidental cell death) or apoptosis (programmed cell death).

Measuring cytotoxicity can prove to be very valuable tool in identifying compounds that might pose certain health risks in humans and animals. Cytotoxicity assays are widely used by pharmaceutical industry to screen for cytotoxicity in compound libraries from initial high-throughput drug screens for unwanted cytotoxicity. This can be of vital importance and prove to be quite indispensable during research phase of developing therapeutic new pharmaceutical products to ensure the safety of end-users. Additionally, understanding the mechanisms involved in cytotoxicity can likewise give researchers a more in-depth knowledge on the biological processes (both normal and abnormal) governing cell growth, cell proliferation and death. Compounds that have cytotoxic effects often compromise cell membrane integrity. Therefore, assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. There are three commonly used methods which can be employed to assess in vitro cytotoxicity (disruption of cell integrity) in cell cultures:

Cell culture refers to removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been establwashed.

Primary culture refers to the stage of culture after the cells are isolated from tissue and proliferated under appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of drug discovery process can save time and money both by eliminating least beneficial NCEs.

Microbial Resistance Mechanism

Huge use of antibiotics in nosocomical environment has lead to the occurrence of multidrug resistance mechanism by microorganism against particular antibiotics (Munita et al., 2016; Zhao et al., 2020). Main operation includes chemical modification, efflux pump through passivation, modification of drug targeting gene and systemic elimination of antibiotics as many of them can form biofilm (Rajput et al., 2018) which can show extreme resistance. Chemical and enzymatic mechanism can include inactivation, activity loss, degradation and derivatizaton of antibiotics chemical group (Liu et al., 2019). Best idea may include production of enzymes or they can also expel antibiotic to efflux pump. Antibiotics drug efflux resistance can be accomplished by drug efflux pump playing an important role in microbial resistance. A different type of efflux pump system has been identified that include lipophilic and hydrophilic efflux system targeting the drug at different chemical properties (Wasaznik et al., 2009; Kumar et al., 2012). Another strategy adopting in resistance mechanism is modification of drug targeting gene. This mechanism makes the changes in drug to lose its target through drug targeting gene modification. Another important resistance mechanism is effectiveness of antibiotics by interfering with the target site. Therefore, bacteria has evolved different mechanism that include target site protection. A bactericidal role played by beta lactum antibiotics in inhibition of mucopeptide synthase and bacterial penicillin binding protein (PBP) in cell wall transpeptidation reaction that prevents the formation of complete call wall and hence dies (Sauvage et al., 2016). Bacteria also developed its own strategies to cope up with the environmental stress that can also include human body. In course of gaining the advantage bacteria needs to compete for nutrients and to keep away from the molecules produced by other competing organism (Zhao et al., 2019). Recently researchers have found the importance of biofilm resistance in bacteria. Bacterial biofilm resistance produce by complex and systematic drug resistance mechanism.

Introduction

The epidemiological risk and hazards to global health security posed by AMR has been reiterating in several World Health Assembly (WHA) declarations and has been prioritized under the Global Health Security Agenda (GHSA). The Indian Ministry of Health & Family Welfare (MoHFW) has also identified AMR as one of the top priorities for collaborative work with WHO and the first National Action Plan on AMR has been implemented (2017-2022). India ranked fifth in antibiotic consumption in food producing animals (ruminants, pigs, and poultry) in 2010. Even though there has been a gradual reduction of 3.6% in antibiotic consumption during 2011-2019 (Koya et al., 2022), with mounting incomes and shifting in dietary patterns in favour of the consumption of animal protein, especially for poultry, the veterinary antibiotic utilization is projected to further rise by 312%, elevating India to the fourth rank by 2030 (Van Boeckel et al., 2015). FAO has already reported some of the peak resistance rates among common commensal and environmental bacteria against antibiotics like penicillin, cephalosporins, erythromycin and co-trimazoles in India (INFAAR). Resistance to the broad spectrum fluoroquinolones and third generation cephalosporin antibiotics is already more than 70% in Escherichia coli, Klebsiella pneumoniae, and, Acinetobacter baumannii and more than 50% in Pseudomonas aeruginosa. Even the microbial resistance to carbapenems, the last-resort antibiotics, is extremely high among gram-negative microorganisms (Sumanth et al., 2016). To overcome these incidences, it is impulsive to understand the microbe and drug interaction in host system.

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

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.

Introduction

Resistance to antibiotics in the bacterial world is a genetically determined phenomenon that was first observed within a few years after the introduction of antibiotics for therapeutic purposes. In Nature, ARGs are believed to have originated about three billion years ago as an intrinsic defense mechanism in antibiotic-producing bacteria (Perry et al., 2016; Iskandar et al., 2022), and subsequently, evolved by complex and quantum jump trajectories to mobilize, diversify and interconnect all ecosystems in the micro-biosphere. Bacterial genomes exhibit plasticity and continue to evolve under the selection pressure of antibiotics and other stressors (Patel, 2016). The ARGs in circulation at present are thus a mixture of ancient and modern counterparts. Recently, the interplay between bacterial metabolism and antibiotic resistance has also been revealed (Zampieri, 2021).

The use of antimicrobials in health management, livestock, and food industry is driving the emergence of resistant microbes at a rapid pace (Wall et al., 2016; Adegoke et al., 2016; Hu et al., 2017). As a result, AR pathogens have spread globally to cause loss of lives, production, productivity, and negatively impact economic growth. Recently, AR pathogens in a large study have been found directly responsible for millions of deaths, and the figures are likely to increase in the coming decades (Murray et al., 2022). The pace of the emergence of AR can however be slowed down by improving human interventions. The global and local AR combat plans implemented during the past few years have enhanced the mass awareness of the pandemic problem (WHO, 2015; FAO, 2016; NAP, GOI, 2017; WHO, 2022; WOAH, 2022). These Action Plans on AR containment encourage avoiding of indiscriminate use of antibiotics for the treatment of sick humans and animals, recommend the ban on the use of antibiotics as growth promoters in food animals, promote AR surveillance and monitoring, and support the ‘One-Health’ strategy and application of the biosecurity principles for containment of the resistant microbes.

Introduction

Antibiotic-resistant bacteria are a major cause of health care-associated infections around the world, and resistance has also emerged in infections in the wider community. Infections caused by multi resistant organisms significantly increase morbidity, mortality, and health care costs. Molecular analyses have revealed that widespread multi resistance has commonly been achieved by the acquisition of preexisting determinants followed by amplification in response to selection. The capture, accumulation, and dissemination of resistance genes are largely due to the actions of mobile genetic elements (MGE), a term used to refer to elements that promote intracellular DNA mobility (e.g., from the chromosome to a plasmid or between plasmids) as well as those that enable intercellular DNA mobility.

Insertion sequences (IS) and transposons (Tn) are discrete DNA segments that areable to move themselves (and associated resistance genes) almost randomly to newlocations in the same or different DNA molecules within a single cell. Other elements, such as integrons (In), use site-specific recombination to move resistance genes between defined sites. As these types of MGE are often present in multiple copies indifferent locations in a genome, they can also facilitate homologous recombination(exchange of sequences between identical or related segments). Intercellular mechanisms of genetic exchange include conjugation/mobilization (mediated by plasmidsand integrative conjugative elements [ICE]), transduction (mediated by bacteriophages), and transformation (uptake of extracellular DNA). Interactions between thevarious types of MGE underpin the rapid evolution of diverse multi resistant pathogens in the face of antimicrobial chemotherapy. Primarily the most important and/ or topical elements in Gram-negative and Gram-positive bacterial species of particular concern clinically, namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (the so-called ESKAPE group) (Rice LB., 2008) as well as Escherichia coli (giving the ESKAPEE group) (Llaca-Díaz J.M. et al.,2012); nonetheless, many of the MGE types described are of course broadly relevant to many other bacterial taxa.

Antibiotics, often referred to as “magic bullets” in the battle against bacteria, stand out as one of the most remarkable medical breakthroughs of the 20th century. Their advent revolutionized the therapeutic landscape, consistently saving millions of lives from bacterial infections (WHO, 2020). Undoubtedly, antibiotics are a blessing to humanity, not merely for their medicinal applications but also for their diverse uses. They have long been employed in various contexts, including animal husbandry and production, serving as preventive measures in numerous underdeveloped and developing nations for decades (Majumder et al., 2020). A range of antimicrobial agents, including antibiotics, antifungals, antivirals, disinfectants, and food preservatives, function to either inhibit microbial growth or eradicate them altogether. Among these, antibiotics, specifically tailored to combat bacterial infections, are the most widely used class of antimicrobials. With their increasing and often improper use, microorganisms have developed antimicrobial resistance (AMR) (Murray et al., 2022).

Antimicrobial resistance is an inevitable evolutionary process observed in all microorganisms, and is driven by genetic mutations aimed at resisting the lethal pressures of selection. Bacteria, adapt to environmental selection pressures by developing resistance to antibacterial drugs, thereby diminishing the efficacy of these treatments (Murray et al., 2022). The widespread use of antibiotics, particularly in developing nations, further flares up the process, leading to significant repercussions such as increased rates of morbidity and mortality (WHO, 2020). The incidence and prevalence of antimicrobial resistant bacterial infections have reached alarming levels in the 21st century, posing a substantial threat to global public health akin to a silent pandemic. Antibiotic resistance knows no borders and can impact individuals of any age or gender, regardless of their location. In its present state, antimicrobial resistance (AMR) stands as one of the foremost threats, not only to global health but also to food security (Murray et al., 2022).

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.

Globally, antimicrobial resistance (AMR) is an important public health and development threat (WHO, 2020). Being one of the top 10 global public health threats, the ever-increasing rates in the occurrence of drug-resistant bacterial infections have become a grave public health concern for the governments (Laxminarayan et al., 2013). Moreover, such infections would be difficult to treat and therefore can lead to worse clinical outcomes (Laxminarayan et al., 2013). Worldwide, each year about 700,000 people die from antimicrobial resistant infections and the mortality figures are projected to reach 10 million/ year by 2050 (O’Neill, 2016). The crude infectious disease mortality rate in India is more than 400 per 100,000 persons and at least 23,000 deaths in adults and 58,000 neonatal deaths per year are caused by AMR bacteria (Laxminarayan and Chaudhury, 2016). A 2017 estimate puts the cost of developing an antibiotic at around US$1.5 billion (Towse, et al. 2017). Meanwhile, industry analysts estimate that the average revenue generated from an antibiotic’s sale is roughly $46 million per year. “That’s tiny and nowhere near the.