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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.