GENERAL CONSIDERATIONS:

The causative agents of human infections diseases belong to 5 major groups of organisms: 1. Bacteria 2. Fungi 3. Protozoa 4. Helminths 5. Viruses The first 3 groups are members of kingdom of PROTISTS, one of the primary biologic sub-divisions along with plants and animals. The protists are distinguished from plants and animals by being either unicellular or relatively simple multicellular. Helminths are complex multicellular which are classified as metazoa within animal kingdom. Helminths and protozoa are commonly called parasites. Viruses are not cells but can replicate only within cells. Cellular organisms differ from viruses as described below. 1) Structure:Cells possess nucleus or nucleoid containing DNA which is surrounded by cytoplasm. Within cytoplasm proteins are synthesized and energy is produced. Viruses have an inner core of genetic material (either DNA or RNA), surrounded by protein coat called capsid, which is made up of sub units called capsomers but no cytoplasm. Hence they (viruses) depend on host cells for protein synthesis and energy generation. 2) Method of Replication:Cells replicate by binary fission in which one parent cell divides into 2 progeny cells retaining its cellular structure. In contrast, viruses disassemble to produce many copies of their nucleic acid and protein and then reassemble into multiple progeny viruses. Viruses must replicate in cells. 3) Nature of the Nucleic Acid:Cells contain both DNA and RNA whereas viruses contain either DNA or RNA but not both.

EUKARYOTES AND PROKARYOTES: Cells are evolved into 2 fundamentally different types, eukaryotic and prokaryotic. Fungi and protozoa are eukaryotic whereas bacteria are prokaryotic. 1- The eukaryotic cell has a true nucleus with multiple chromosomes surrounded by a nuclear membrane and uses mitotic apparatus to ensure equal allocation of chromosomes to progeny cells. The nucleoid of prokarytoic cell consists of a single circularmolecule of loosely organized DNA lacking a nuclear membrane and mitotic apparatus. 2. Eukaryotic cells contain organelles, like mitochondria and lysosomes and larger (80 S) ribosomes having 60S and 40S subunits whereas prokarytoes contain no organelles and smaller (70S) ribosomes with 50S and 30S subunits. Most prokaryotes contain a rigid external cell wall that contains peptidoglycan, a polymer of amino acids and sugars as its unique structural unit. On the other hand eukaryotes do not contain peptidoglycan. 3. Eukaryotic cell membrane contains sterols whereas no prokaryote (except the wall-less mycoplasma) has sterols in its membranes. STRUCTURE OF BACTERIAL CELL: Bacteria are probably the simplest and the smallest living organisms possessing cellular organization. Depending upon shape they are classified into 3 basic groups. 1. Cocci (single coccus) spherical in form e.g. shapes cluster staphylococci or streptococci (chain) pair. 2. Bacilli (single bacillus) rod shaped (a) with square ends, (b) with rounded ends, (c) club shaped, (d) fusiform, (e) coma shaped. 3. Spirochetes or spirilla (spiral, cork or screw shaped) (a) relaxed coiled (b) tightly coiled. Some bacteria are variable in shape in shape and they are called pleomorphic (many shaped). In addition to shapes, the arrangement of bacteria is also important. For example certain cocci occur in pairs (Diplococci) some in chains (streptococci) and other in grapelike bunches (staphylococci) SIZE:Bacteria range in size from about 0.2 to 5 m. The smallest bacteria are about the same size as largest viruses poxviruses. The longest bacterial rod approaches the size of some yeast and human red cell i.e. 7 m.

STRUCTURE CELL WAL:The function of the cell wall is to support under lying plasma membrane which is subject to an internal osmotic pressure of about 5 atmospheres in G-negative and about 20 atmospheres in G-positive bacteria. The cell wall is multilayered structure located external to cytoplasmic membrane. Plasma membrane and cell wall together comprise envelope. It is composed of an inner layer of peptidoglycan, surrounded by an outer layer (outer membrane) that varies in thickness and chemical composition. The peptidoglycan provides structural shape and maintains characteristic shape of cell. The cell wall of Gram Positive bacteria differs from G-Negative bacteria as under. The peptidoglycan layer is much thicker in Gram positive (50-100 molecules thick) than in Gram negative bacteria (1-2 molecules thick). Some Gram positive bacteria have a layer of teichoic acid on the outside of the peptidoglycan. Whereas G-negative bacteria do not have this layer. In contrast, the G-negative bacteria have a complex outer layer (out membrane) consisting of lipopolysaccharide, lipoprotein and phospholipid. There is no outer Membrane present in G-positive bacteria. The out membrane in gram negative bacteria contains porins (pore molecules) through which hydrophilic drugs pass more readily. Lying between the outer membrane layer and the cytoplasmic membrane in gram negative bacteria is the periplasmic space. This is the site, in some bacteria, of enzymes that degrade penicillins and other -lactam drugs. The cell wall has other properties as well. e.g. In gram negative bacteria:i. The cell wall conatins endotoxins, alipopolysacharide ii. Its polysaccharides and proteins are antigens, that are useful in lab. Identification and ii- Its porin proteins play a role in regulating the passage of molecules into the cell. CELL WALL of ACID FAST BACTERIA: Mycobacteria have unusual cell wall resulting in their inability to be Gram-stained. These are called Acid-Fast since they resist decolonization with acid alcohol after being stained with carbofuchsin. The property is related to high concentration in the cell wall of lipids called mycolic acids.

MESOSOME:This cytoplasmic membrane invagination is important during cell division, when it functions as the origin of transverse septum that divides the cell in half and as the binding site of the DNA which will become the genetic material of each daughter cell. CAPSULE:Capsule is a gelatinous layer covering the entire bacterium. It is composed of polysaccharide, except in anthrax bacillis, which ahs a capsule of polymerized D-glutamic acid. Capsule is important for 4 reasons. 1. It is determinate of virulence of many bacteria, since it limits the ability of phagocytes to engulf the bacteria. 2. Specific identification of an organism can be made by using antiserum against the capsular polysaccharide. 3. Capsular polysaccharide are used as antigen in certain vaccines, since they are capable of eliciting protective antibodies. 4. The capsule may play role in the adherence of bacteria to human tissue, which is important initial step in causing infection. SPECIALIZED STRUCTRE OUTSIDE CELL WALL : PILI (Fimbriae): are hair-like filaments extending from the cell surface. They are found mainly on G-negative bacteria. They have 2 important functions. i. They help in the attachment of bacteria to specific receptors on human cell surface which is necessary step in initiation of infection for some organisms. ii. A specialized kind of pilus, the sex pilus forms the attachment between the male (donor) and the female (recipient) bacteria during conjugation. FLAGELLA:Flagella are long, whiplike appendages that propel the bacteria towards food or away from harmful substances, the process is called chemotaxis. SPORES:Highly resistant structures are formed in response to adverse conditions by 2 genera. Bacillus and Clostridium.

GROWTH OF BACTERIA Growth Cycle: Bacteria reproduce by binary fission a process by which a parent cell divides to form two progeny cells. As one cell gives rise to two daughter cells, bacteria are said to undergo exponeutial growth (Logrithmic growth) i.e. Number of cells Exponential 1 20 2 21 4 22 8 23 16 24

The doubling time for bacteria ranges from as little as 20 minutes (E. Coli) to more than 24 hr (Mycobacterium tuberculosis). For example E. Coli E. Coli after 3hrs 1000

after 7 hrs over one million

The doubling time varies not only with species but also with amount of nutrient, pH, temperature and so many other factors. The growth curve fig:

1. In lag phase vigorous metabolic activity occurs but no division takes place. 2. In log phase rapid cell division occurs 3. In stationary phase, depletion of nutrients or production of toxic substance case growth to show until the number of new cell produced balance the cells that die. 4. Death phase is marked by a decline in number of viable bacteria.

PATHOGENESIS: A microorganism is a pathogen if it is capable of causing disease. Some organisms are frequently pathogens, whereas other cause disease rarely. Opportunistic are those, which rarely if ever cause disease in immune competent people but can cause serious disease in those individuals who have reduced defense. VIRULENCE:Virulence is quantitative measure of pathogenecity. In the context of virulence the 50 % lethal dose (LS50) is the number of organisms needed to kill half of the hosts. In the context of 50 % infections dose (ID50) is the number of organisms needed to cause infection if half the hosts. EPIDEMIC:An infection is epidemic if it occurs much more frequently than usual. It is pandemic if it has a worldwide distribution. An endemic infection is constantly present at a low level in a specific population. GRAM STAIN: This staining procedure is the most important in microbiology. It was developed in 1884 by Danish Physician CHRISTIAN GRAM. It separates most bacteria into 2 groups: Gram Positive bacteria which stain blue, and the gram negative bacteria which stain red. The gram stain involves the following 4-step procedure 1- Crystal violet dye all cells blue. 2. The iodine solution ( mordant ) is added to form a crystal violet-iodine complex; all cells continue to appear blue. 3. The organic solvent, such as acetone or ethanol extracts the blue dye complex from the lipid rich, thin walled gram-negative bacteria to a greater degree than from the lipid poor, thick walled gram positive bacteria. The gram negative organisms appear colourless; the gram positive bacteria remain blue. 4- The red dye safranin stains the decolorized gram negative cells red, the gram positive bacteria remain blue.

Importance:Identification of bacteria for better choice of antibiotic. NOTE:Immuno compromise:Having the immune response attenuated by administration of immuno suppressive agents, by irradiation by malnutrition and by some disease process. Cytoplasmic Membrane:Just inside the peptidoglycan, lies the cytoplasmic membrane which is composed of phospholipid bilayer similar in microscopic appearance to that of eukaryotic cells. They are chemically similar but eukaryotic membrane contains sterols whereas prokaryotes generally do not the only prokaryotes that have sterols in their membrane are mebers of the genus mycoplasma membrane has four important functions: I. Active transport of molecules into cells II. Energy generation by oxidative phosphoriylation III. Synthesis of precursors of cell-wall IV. Secretion of enzymes and toxins. CYTOPLASM: It has two distinct areas when seen under electron microscope: 1. An orphous matrix that contains ribosomes, nutrients, granules, metabolites and ions. 2. An inner nucleoid region composed of DNA. A. Ribosome: Bacterial ribosomes are the sites of protein synthesis as in eukaryotic cells but they differ from eukaryotic ribosomes in size and chemical composition. Bacterial ribosomes are 70 S in size having 50 S and 30 S in size, with 60 S and 40 S subunits. The difference in both ribosomal RNAs and protien constitute the basis of the selective action of several antibiotics that inhibit bacterial but not human protein synthesis.

B. Granules:Granules serve as storage area for nutrients and stain characteristically with certain dyes. C. Nucleoid:It is the area of cytoplasm in which DNA is located. The DNA of prokaryotes is a single, circular molecule that has molecule of approximately 2 x 109 and contains about 2000 genes. The nucleoid contains no nuclear membrane, no mitotic apparatus and no his tones (simple protein) D. Plasmids:Plasmids are extra chromosomal, double-stranded circular DNA molecules that are capable of replicating independently of the bacterial chromosome. Although they are extra-chromosomal, they can be integrated into the bacterial they can be integrated into the bacterial chromosome. Plasmid can be 1. Transmissible:Plasmids can be transferred from one cell to another by conjugation. They are large because they contain about a dozen genes responsible for synthesis of sex pilus and enzymes required for transfer. 2. Nontransmissible:Nontransmissible are small since they do not contain transfer genes. Plasmids occur in both Gram positive and the genes for the Gram negative bacteria following functions and structures of medical importance are carried by plasmids. 1. Antibiotic resistance that is mediated by variety of enzymes. 2. Resistance to heavy metals e.g. Hg. 3. resistance to UV light which is mediated by DNA repair enzymes. 4. pili which mediate adherance of bacteria to epithelial cells. 5. Oxotoxins:A toxic substance formed by bacteria that is found outside the bacterial cell or free in the culture medium.

Other plasmid-encoded products of interest are 1. bacteriocins toxins or enzymes produced by certain bacteria and one lethal to other bacteria. 2. nitrogen fixation enzymes in Rhizopodium in the roots of legumes. 3. tumour caused by agrobacterium in plants. 4. certain antibiotics produced by streptomyces. 5. a variety of degradative enzymes. E-Transposons:E-transposons are pieces of DNA that more readily from one place to another, either within or between the DNAs of bacteria, plasmids and bacteriophages. They are named jumping genes (nick name). They can code for drug resistance enzymes, toxins or drug metabolizing enzymes.