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CHAPTER I INTRODUCTION Infections caused by microorganisms especially bacteria and viruses are the major threat to the human beings especially children. Recent reports revealed that 10% of the worldwide burden of morbidity and mortality relates to respiratory tract infections as they kill an estimated 10 million people annually. The majority of these cases are children under five years old in developing countries [1]. Upper respiratory tract infections account for more visits to physicians than any other type of infectious disease. Lower respiratory tract infection is a common cause of hospital admission [2, 3]. Upper respiratory tract infections may move downwards and result in more severe infections of the lower respiratory tract, such as pneumonia or bronchiolitis [4]. This progression towards more severe respiratory tract infection is of significant concern in developing countries where pneumonia contributes substantially to childhood death [5]. Different microorganisms are recognized as important etiologic agents of upper respiratory tract infections. Staphylococcus aureus, Proteus, Klebsiella, and Pseudomonas were reported to be the most important causes of chronic suppurative otitis media [6]. Streptococcus pneumoniae, Haemophilus influenzae, group A Streptococci and Staphylococcus aureus are often isolated from cases of sinusitis [7]. Streptococcus pyogenes is by far the most clinically important etiologic agent of bacterial pharyngitis [8]. Infecting organisms of tonsillitis are identical in type and incidence to those causing pharyngitis with the addition of Staphylococci, S. pneumoniae and Haemophilus sp. [9]. S. pneumoniae is the most common bacterial pathogen in lower respiratory tract infections [10]. Lower respiratory tract infections (LRTIs) are among the most dreadful diseases affecting humans worldwide. They are important causes of morbidity and mortality for all age groups, and each year approximately seven million people die as a direct consequence of acute and chronic respiratory infection [11]. Lower respiratory tract infections (LRTI’s) may be defined as those infections presenting with symptoms including cough, expectoration, dyspnoea, wheeze and or chest pain or discomfort usually for a period ranging from 1-3 weeks. Acute manifestations of lower respiratory tract infections that may or may not involve lungs include acute bronchitis, bronchiolitis, influenza, community–acquired pneumonia either with or without radiological evidence, acute exacerbation of Chronic obstructive pulmonary disease (COPD) and acute exacerbation of broncheictasis [12]. Lower respiratory tract infections, including acute and chronic bronchitis, pneumonia and pneumonia in immune-compromised hosts, are among the most common major infectious illness [13]. LRTI’s have been attributed to account for almost 20% mortality among the infectious disease deaths in India as reported by World Health Organization [14]. It has been reported that diagnostic laboratories and clinical microbiologists have a critical role to play in the diagnosis and management of LRTI’s [15, 16]. Lower respiratory tract infections include infection of trachea, bronchi and lungs. It usually occurs when infecting organisms reach the airway of pulmonary parenchyma by bypassing the mechanical and other nonspecific barriers of the upper respiratory tract. Infection may result from inhalation of infectious aerosols, aspiration of oral or gastric contents or by heterogeneous spread [17]. Most common lower respiratory infections are acute tracheobronchitis, acute exacerbations of chronic bronchitis and pneumonia [18] and common causative agents are Streptococcus pneumoniae, Haemophilus influenzae, Acinetobacter baumannii, Enterococcus sp, Klebsiella pneumoniae etc. Current knowledge of the organisms that cause LRTIs and their antibiotic susceptibility profiles are therefore necessary for the prescription of appropriate therapy. Studies have been conducted to determine the microbial agents of human respiratory tract infections, the effect of age and gender on the prevalence of LRTIs, and the susceptibility pattern of bacterial isolates [19]. Acinetobacter baumannii is a common gram-negative bacterium, which is an opportunistic nosocomial pathogen [20]. A. baumannii is of increasing medical importance due to its ability to colonise quickly and give resistance too many antibiotics [21]. Due to the increasing resistance of Acinetobacter sp. infections have become increasingly difficult to treat [22]. There have also been many reports of resistance of infections in military personnel [23] and have shown a wide variety in types of infection caused by Acinetobacter [24]. Over the last 30 years the types of infection that Acinetobacter baumannii cause has increased not only in hospitals but also in community acquired infections [25]. During the last two decades, A. baumannii has become a pathogen of increased clinical importance due to its remarkable ability to cause outbreaks and its ability to acquire resistance to almost all available antibiotics, including the carbapenems (such as imipenem and meropenem) [26, 27]. Carbapenems are the drugs of choice for the treatment of serious nosocomial infections caused by A. baumannii [28, 29]. Carbapenem-resistant A. baumannii strains have now emerged around the world [30, 31, 32]. To better understand the epidemiology and in particular the mode of spread of A. baumannii, a number of molecular typing systems have been developed, including PCR-based methods such as RAPD analysis [33], integrase gene PCR [34], infrequent-restriction-site PCR [35], ribotyping [36, 37], amplified fragment length polymorphism (AFLP) analysis [38] and PFGE [39]. All of these methods rely on the generation of a distinct pattern of DNA ―fingerprint that is usually visualized by ethidium bromide staining or nucleic acid hybridization. The so-called comparative typing systems, i.e., methods that depend on comparisons of DNA fragment patterns on gels, such as PFGE and RAPD analysis, are told to be well suited for local outbreak investigation. Klebsiella sp. has been reported as opportunistic, worrisome nosocomial and communityassociated pathogens [40]. These bacteria have become important pathogens in nosocomial infections [41] which have been well documented in United States and India [42]. Epidemic and endemic nosocomial infections caused by Klebsiella species are leading causes of morbidity and mortality [43]. It is very common among inpatients admitted in ICUs for various ailments. The widespread emergence of multidrug resistance among bacterial pathogens has become one of the most serious challenges in clinical therapy [44]. RAPD-PCR is one of the most rapid and simple methods that generate fingerprints and it can be applied to detect polymorphism in a wide variety of organisms. In RAPD-PCR, random primer sequences may be used in organisms where a specific genome sequence is not known. Random parts of the organisms genome are formed, which are expected to be identical among related species, and so similar banding patterns should be produced in gel electrophoresis. This technology is proving to be quite useful in typing strains of bacteria involved in respiratory tract diseases [45]. Medicinal plants are the primary source of medicine for the treatment of human diseases in many rural areas of the developing countries [46]. Medicinal plants have been used in treating various diseases in alternative medicines such as siddha, ayurveda, unani from time immemorial. The medicinal value of the plant is due to the presence of various bioactive chemical constituents such as alkaloids, tannins, flavonoids and phenolic compounds [47]. Multiple drug resistance in pathogenic bacteria has developed due to the use and misuse of antibiotics. Therefore scientists have started looking for new antibiotics and also there is a strong need to develop antimicrobial drugs from medicinal plants to control the infectious and emerging strains of pathogenic microorganisms [48]. In India, the use of different parts of several medicinal plants to cure unambiguous ailments has been in practice from prehistoric times. In early periods, human beings have been dependent on plants for their care and other needs. According to a WHO survey, 70–80% of the world population is dependent on plant based medicines. Herbalism uses the whole plant or part of the medicinal plant such as leaves, flowers, fruits, stems or root rather than separating the active agent. Plant contain active constituents that work together synergistically with no/low side effects, which may occur if separated components are used. Ministry of Health and Family Welfare, Government of India has established the Department of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homeopathy (AYUSH). Traditional medicine system has expanded global importance. Hence, a thorough knowledge on their organic constituents and trace elements contents to its role in treating ailment is called for in providing safe and effective medicinal plants [49, 50, 51]. Antioxidants are substances that neutralize free radicals or their action [52]. Nature has endowed each cell with adequate protective mechanisms against any harmful effects of free radicals: In every cell superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, thioredoxin, thiols and disulfide bonding are buffering systems. α- Tocopherol (Vitamin E) is an essential nutrient which functions as a chain-breaking antioxidant which prevents the propagation of free radical reactions in all cell membranes in the human body. Ascorbic acid (vitamin C) is also part of the normal protecting mechanism. Carotenoids, flavonoids and related polyphenols, α-lipoic acid and glutathione are other non enzymatic antioxidants. At different stages antioxidants are capable of neutralizing free radicals or their actions. They act at the levels of prevention, interception and repair. Preventive antioxidants attempt to stop the formation of ROS.These include superoxide dismutase (SOD) that catalyses the dismutation of superoxide to H2O2 and catalase that breaks it down to water [53]. The relation between free radicals and disease can be explained by the concept of ‘oxidative stress’ [54]. AIM AND OBJECTIVES The nosocomial lower respiratory tract pathogens are opportunistic and can remain alive on life less interface especially in hospital equipments for many days. They are getting resistant even to the stronger classes of antibiotics such as carbopenems. They are becoming very difficult to treat and also the situation demands to look for alternate drug resources to combat these pathogens. Considering these facts, the present study was carried out with the following objectives: To isolate lower respiratory tract infection causing bacterial pathogens from suspected patients from various clinical laboratories of Kanyakumari district, TamilNadu. To identify the pathogens using various morphological and biochemical tests. To confirm the identity of the bacterial pathogen using 16srRNA gene sequencing. To Study the antibiotic sensitivity pattern of bacterial pathogens against commercial antibiotics by Disc diffusion method. To identify the genetic polymorphism among the bacterial isolates using PCR-RAPD technique. To analyze the phytochemical constituents of selected medicinal plants. To identify the phytochemical compounds present in the selected medicinal plants using UV-Visible, FT IR and GC MS analysis. To assess the antimicrobial activity of the various solvent extracts of the selected medicinal plants against the previously isolated lower respiratory tract pathogens. To study the antioxidant potential of the selected medicinal plants using the superoxide dismutase activity. To carry out the docking analysis of the phytochemical constituents with the bacterial target proteins using bioinformatics tools.