Microbiology Unit 3: Eukaryotes & Microorganism Control PDF

Unit 3 Structure of Eukaryotes & Control of Microorganisms *Characteristics, Morphology, Reproduction, Physiology and Pathogenicity Fungi Algae...

Unit 3 Structure of Eukaryotes & Control of Microorganisms *Characteristics, Morphology, Reproduction, Physiology and Pathogenicity Fungi Algae Protozoa * Control of Microorganisms Physical control Chemical control Antibiotics  Unit 3 - FUNGI  Fungi - Importance of fungi in various field applications  Morphology of fungi  Structural characteristics and ecological association of fungi  Classification of fungi  Sexual and Asexual Reproduction of fungi  Cultivation of fungi  Preservation techniques of fungi  Fungal toxins FUNGI  Microbiologists use the term fungus [pl., fungi; Latin fungus, mushroom] to describe eucaryotic organisms that are spore-bearing, have absorptive nutrition, lack chlorophyll, and reproduce sexually and asexually.  Scientists who study fungi are mycologists [Greek mykes, mushroom, and logos, science], and the scientific discipline devoted to fungi is called mycology.  The study of fungal toxins and their effects is called mycotoxicology, and the diseases caused by fungi in animals are known as mycoses (s., mycosis).  According to the universal phylogenetic tree, fungi are members of the domain Eucarya.  Morphological, biochemical and molecular phylogenetic analyses demonstrate that the Fungi constitute a monophyletic group.  They are sometimes referred to as the true fungi or Eumycota [Greek eu - true, and myke - fungus].  DISTRIBUTION  Fungi are primarily terrestrial organisms, although a few are freshwater or marine.  They have a global distribution from polar to tropical regions.  Many are pathogenic and infect plants and animals.  Fungi also form beneficial relationships with other organisms.  For example, the vast majority of vascular plant roots form associations (called mycorrhizae) with fungi.  Fungi also are found in the upper portions of many plants.  These endophytic fungi affect plant reproduction and palatability to herbivores.  Lichens are associations of fungi and photosynthetic protists or cyanobacteria.  IMPORTANCE  Fungi are important to humans in both beneficial and harmful ways.  Fungi act as decomposers, a role of enormous significance. They degrade complex organic materials in the environment to simple organic compounds and inorganic molecules.  Fungi are a major cause of disease. Plants are particularly vulnerable to fungal diseases because fungi can invade leaves through their stomates.  Fungi also cause many diseases of animals and humans.  Fungi, especially the yeasts, are essential to many industrial processes involving fermentation. Eg: Making of bread, wine, and beer.  Fungi also play a major role in the preparation of some Cheeses, Soy sauce, and Sufu;  In the commercial production of many organic acids (Citric, gallic) and certain drugs (Ergometrine, Cortisone); and  In the manufacture of many antibiotics (Penicillin, Griseofulvin) and the immunosuppressive drug cyclosporin. Some Mycotoxicoses Produced by Fungal Mycotoxins in Domestic Animals  STRUCTURE  The body or vegetative structure of a fungus is called a thallus.  It varies in complexity and size, ranging from the single-cell microscopic yeasts to multicellular molds, macroscopic puffballs, and mushrooms.  The fungal cell usually is encased in a cell wall of chitin.  Chitin is a strong but flexible nitrogen containing polysaccharide consisting of N- acetylglucosamine residues  YEAST  A yeast is a unicellular fungus that has a single nucleus and reproduces either asexually by budding and transverse division or sexually through spore formation.  Each bud that separates can grow into a new yeast, and some group together to form colonies.  Yeast cells are commonly spherical to egg shaped.  They lack flagella but possess most of the other eucaryotic organelles.  MOLDS  The thallus of a mold consists of long, branched, threadlike filaments of cells called hyphae that form a mycelium, a tangled mass or tissue like aggregation of hyphae.  In some fungi, protoplasm streams through hyphae, uninterrupted by cross walls. These hyphae are called coenocytic or aseptate.  The hyphae of other fungi have cross walls called septa with either a single pore or multiple pores that enable cytoplasmic streaming. These hyphae are termed Mutiporate septate. hyphae diagram  Hyphae are composed of an outer cell wall and an inner lumen, which contains the cytosol and organelles.  A plasma membrane surrounds the cytoplasm and lies next to the cell wall.  The filamentous nature of hyphae results in a large surface area relative to the volume of cytoplasm.  This makes adequate nutrient absorption possible. FUNGAL HYPHAL MORPHOLOGY DIMORPHIC FUNGI  Many fungi, especially those that cause diseases in humans and animals, are dimorphic - that is, they have two forms.  Dimorphic fungi can change from the yeast (Y) form in the animal to the mold or mycelial form (M) in the external environment in response to changes in various environmental factors (nutrients, CO2 tension, oxidation-reduction potentials, temperature). This shift is called the YM shift. Some Medically Important Dimorphic Fungi  NUTRITION AND METABOLISM  Fungi grow best in dark, moist habitats and are found wherever organic material is available.  Most fungi are saprophytes, securing their nutrients from dead organic material.  They are chemoorganoheterotrophs and use organic compounds as a source of carbon, electrons, and energy.  Glycogen is the primary storage polysaccharide in fungi.  Most fungi use carbohydrates (preferably glucose or maltose) and nitrogenous compounds to synthesize their own amino acids and proteins.  Fungi usually are aerobic. Some yeasts, however, are facultatively anaerobic and can obtain energy by fermentation. Obligately anaerobic fungi are found in the rumen of cattle.  Many fungal fermentations are of industrial importance, such as the production of ethyl alcohol in the manufacture of beer and wine.  ECOLOGY OF FUNGI  Fungi, together with bacteria are the principal decomposers in the biosphere  Fungi are virtually the only organisms capable of breaking down cellulose and lignin  Fungi have entered into fascinating symbioses with a variety of life forms * Obligate symbiosis – Essential for fungus survival * Facultative symbiosis – Non essential ECOLOGY OF FUNGI Types of symbioses -Pathogens and parasites benefit at the expense of their host -Commensals benefit one partner but do not harm or benefit the other -Mutualistic relationships benefit both partners ECOLOGY OF FUNGI Endophytic fungi live in the intercellular spaces inside plants ►Some fungi protect their hosts from herbivores by producing toxins ► Rye grass is more resistant to aphid feeding in the presence of endophytes  LICHENS  Lichens are the association between specific ascomycetes (a fungus) and certain genera of either green algae or cyanobacteria.  In a lichen, the fungal partner is termed the mycobiont and the algal or cyanobacterial partner, the phycobiont.  In the past the lichen symbiosis was considered to be a mutualistic interaction.  It recently has been found that a lichen forms only when the two potential partners are nutritionally deprived.  In nutrient-limited environments, the relationship between the fungus and its photosynthetic partner has coevolved to the point where lichen morphology and metabolic relationships are extremely stable.  The characteristic morphology of a given lichen is a property of the association and is not exhibited by either symbiont individually.  Because the phycobiont is a photoautotroph-dependent only on light, carbon dioxide, and mineral nutrients—the fungus can get its organic carbon directly from the alga or cyanobacterium.  The fungus often obtains nutrients from its partner by projections of fungal hyphae called haustoria, which penetrate the phycobiont cell wall.  It also uses the O2 produced during phycobiont photophosphorylation in carrying out respiration.  In turn the fungus protects the phycobiont from high light intensities, provides water and minerals to it, and creates a firm substratum within which the phycobiont can grow protected from environmental stress.  The invasive nature of the fungal partner is why lichens are considered parasitic relationships.  MYCORRHIZAE  Mycorrhizae (derived from the Greek “fungus root”) are mutualistic relationships that develop between most plants and a limited number of fungal species.  Both partners in mutualistic relationships are dependent on the activities of the other and as such have coevolved.  In this case, fungi colonize the roots of about 80% of all higher plants as well as ferns and mosses and they use photosynthetically derived carbohydrate provided by their host.  In return, they provide a number of services for their plant hosts, including enhanced nutrient uptake.  Arbuscular (endo) mycorrhizae -Hyphae penetrate the root cell wall -By far the most common (70% of all species) -Fungal partners are Glomeromycetes  Ectomycorrhizae -Hyphae surround but do not penetrate the root cells -Most hosts are forest trees (pines,oaks) -Fungal partners are mostly Basidiomycetes Advantages Of Mycorrhizae CLASSIFICATION OF FUNGI ZYGOMYCETES Lower fungi Broad, septate hyphae Asexual spores - Sporangiospores: present within a swollen sac- like structure called Sporangium ZYGOMYCETES Sexual spores - Zygospore: a resting, thick walled cell in between hyphae e.g. Rhizopus, Mucor ASCOMYCETES Includes both yeasts & filamentous fungi Narrow, septate hyphae Asexual spores are called conidia borne on conidiophore  ASCOMYCETES Sexual spores called ascospores are present within a sac like structure called Ascus. Several asci may be seen within a fruiting body as seen in Penicillium, Aspergillus Each ascus has 4 to 8 ascospores.  BASIDIOMYCETES Sexual fusion results in the formation of a club shaped organ called base or basidium which bear spores called basidiospores DEUTEROMYCETES OR FUNGI IMPERFECTI Grow as molds as well as yeasts. Asexual stage – conidia e.g. Candida, Cryptococcus REPRODUCTION  Reproduction in fungi can be either asexual or sexual.  Asexual reproduction is accomplished in several ways: 1. A parent cell can undergo mitosis and divide into two daughter cells by a central constriction and formation of a new cell wall. Transverse fission forming new cell wall 2. Mitosis in vegetative cells may be concurrent with budding to produce a daughter cell. This is very common in the yeasts. 3. The most common method of asexual reproduction is spore production. Asexual spore formation occurs in an individual fungus through mitosis and subsequent cell division.  There are several types of asexual spores, each with its own name: a. A hypha can fragment (by the separation of hyphae through splitting of the cell wall or septum) to Hyphal fragmentation form cells that behave as spores. resulting in arthroconidia (arthrospores) These cells are called arthroconidia or arthrospores. Terminal chlamydospore b. If the cells are surrounded by a thick wall before separation, they are called Chlamydospore within a hyphae chlamydospores. c. If the spores develop within a sac (sporangium; pl., sporangia) at a hyphal tip, they are called sporangiospores. d. If the spores are not enclosed in a sac but produced at the tips or sides of the hypha, they are termed conidiospores. e. Spores produced from a vegetative mother Blastospores cell by budding are called blastospores Vegetative mother cell  Sexual reproduction in fungi involves the fusion of compatible nuclei. Depending on the species, sexual fusion may occur between haploid gametes, gamete-producing bodies called gametangia, or hyphae.  Sometimes both the cytoplasm and haploid nuclei fuse immediately to produce the diploid zygote.  Usually, however, there is a delay between cytoplasmic and nuclear fusion. This produces a dikaryotic stage in which cells contain two separate haploid nuclei (N N), one from each parent.  After a period of dikaryotic existence, the two nuclei fuse and undergo meiosis to yield spores. For example, in the zygomycetes the zygote develops into a zygospore; in the ascomycetes, an ascospore; and in the basidomycetes; a basidiospore. Zygomycetes Ascomycetes Basidiomycetes  Fungal spores are important for several reasons.  The spores enable fungi to survive environmental stresses such as desiccation, nutrient limitation, and extreme temperatures, although they are not as stress resistant as bacterial endospores.  Because they are often small and light, spores can remain suspended in air for long periods.  Fungal spores often spread by adhering to the bodies of insects and other animals.  The size, shape, color, and number of spores are useful in the identification of fungal species. CULTIVATION OF FUNGI  Potato Dextrose Media, Sabouraud’s Dextrose media, Rose Bengal Media medium  Batch/continuous culture  Grown under both aerobic and anaerobic conditions  Long periods (5-50 days)  Tem 20-40 C  Colonial Morphology of Fungi CryptococcuCandida Trichophyto T. menta- Wangiella Aspergillu s s albicans n tonsurans grophytes dermatitidi neoformans s fumigatus PRESERVATION TECHNIQUES OF FUNGI  Stock cultures are aseptically transferred at appropriate intervals to fresh medium and incubated, then stored at 4°C until they are transferred again  Many labs use “agar slants;” care has to be taken to avoid contamination  Microorganisms require special storage methods in order to ensure optimal long-term viability and genetic stability. Preservation method can be assigned to one of the following groups: 1. Metabolically inactive preservation methods 2. Metabolically active methods cryocan liquid nitrogen container lyophilizer  METABOLICALLY INACTIVE PRESERVATION TECHNIQUES Advantages: Viable Cryopreservation up to 5 year  Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as −196 °C Freezing and low temperature storage in or above liquid nitrogen (- 196°C)  Freezing and low temperature storage below -70°C Drying  Preservation by spin freeze-drying  Preservation by vacuum drying  Metabolically active preservation techniques CryptococcuCandida Trichophyto T. menta- Wangiella Aspergillu s s albicans n tonsurans grophytesdermatitidi neoformans s fumigatus  FUNGAL TOXINS Rot disease of Peanut caused by Aspergillus niger Aspergillus niger  Aflatoxin B1 Aspergillus species Hepatocellular carcinoma (HCC), or liver cancer, is the third leading cause of cancer deaths worldwide [World Health Organization (WHO) 2008], with roughly 5,50,000-6,00,000 new HCC cases globally each year (Ferlay et al. 2004). Aflatoxin exposure in food is a significant risk factor for HCC (Wild and Gong 2010). IMPORTANCE OF FUNGI  Pharmaceuticals- Penicillin, Enzymes  Agriculture- Bt cotton, Biofertilizer/Biocontrol (T. viride)  Baking industry -  Fermentation- Alcohol  Pathology- Animals& Plants  Genetic engineering- Yeast artificial chromosomes (YACs) -Yeast two-hybrid system  ALGAE The algae (singular, alga), many of which are unicellular microorganisms. These organisms are ubiquitous; many live in aquatic environments but many also thrive as terrestrial and subterranean algae. Algae contain chlorophyll and are photosynthetic. They differ from other green plants in having simple reproductive structures for sexual reproduction. They range from microscopic unicellular forms comparable in size to bacteria to seaweeds that may grow to many feet in length. The study of this unique group of organisms is called phycology.  OCCURRENCE They occur in great abundance in the oceans, seas, salt lakes, freshwater lakes, ponds, and streams. Many are found in damp soil, on rocks, stones, and tree bark, and on other plants and animals. Algae are found where there are sufficient light, moisture, and simple nutrients to sustain them Small aquatic forms make up a large part of the free-floating microscopic life in water called plankton, which is the principal food for aquatic animals, including such large ones as whales. (Plankton is generally considered to be composed of both algae and microscopic animal forms. Phytoplankton is made up of plants, i.e., algal forms, and zooplankton is composed of animal organisms.  - Four types of algae Unicellular Colonial Filamentous multicellular Chlamydomo Volvox Spirogyra.Ulva sp nas  MORPHOLOGY Many species occur as single cells that may be spherical, rod-shaped, club-shaped, or spindle-shaped. Others are multicellular and appear in every conceivable form, shape, and degree of complexity, including membranous colonies, filaments grouped singly or in clusters with individual strands that may be branched or unbranched, and tubes. Algal cells are eucaryotic. Cell wall - Thin and rigid. The cell walls of many algae are surrounded by a flexible, gelatinous outer matrix secreted through the cell wall, reminiscent of bacterial capsules. As the cells age, the outer matrix often becomes pigmented and stratified. Algae contain a discrete nucleus. Other inclusions are starch grains, oil droplets, and vacuoles  ALGAL PIGMENTS The chloroplasts of different divisions of algae containing similar pigments appear to have similar thylakoid arrangements. Chloroplast ultrastructure and pigment chemistry have been used as markers for algal phylogeny. Chlorophylls - There are five chlorophylls; a, b, c, d, and e. Carotenoids - There are two kinds of carotenoids: carotenes and xanthophylls. Carotenes are linear, unsaturated hydrocarbons, and xanthophylls are oxygenated derivatives of these. Biloproteins (Phycobilins) - These are water-soluble pigments, whereas chlorophylls and carotenoids are lipid-soluble. There are two kinds of phycobilins: phycocyanin and phycoerythrin. The proportion of one kind of pigment to another can vary considerably with changes in environmental conditions.  MOTILITY The motile algae, also called the swimming algae, have flagella occurring singly, in pairs, or in clusters at the anterior or posterior ends of the cell. It will suffice for this discussion to mention three types: whiplash (cylindrical and smooth); tinsel (cylindrical and with hairlike appendages); and ribbon, or straplike. Some algae have no means of locomotion and are carried about by tides, waves, and currents.  REPRODUCTION Algae may reproduce either asexually or sexually. ASEXUAL REPRODUCTION ⁎ Asexual reproductive processes in algae include the purely vegetative type of cell division by which bacteria reproduce. ⁎ A new algal colony or filament may even start from a fragment of an old multicellular type from which it has broken. ⁎ However, most asexual reproduction in algae is more complex than this and involves the production of unicellular spores, many of which, especially in the aquatic forms, have flagella and are motile; these are called zoospores. ⁎ The nonmotile spores, or aplanospores, are more likely to be formed by the terrestrial types of algae. However, some aplanospores can develop into zoospores. Sexual reproduction: ⁎ There is a fusion (conjugation) of sex cells, called gametes. to form a union in which "blending" of nuclear material occurs before new generations are formed. ⁎ The union of gametes forms a zygote. ⁎ If the gametes are "identical," i.e., if there is no visible sex differentiation, the fusion process is isogamous. ⁎ If the two gametes are unlike, differing in size (male and female), the process is heterogamous Classification of algae Division Examples Chlorophyta Chlamydomonas, Volvox Euglenophyta Euglena Pyrrophyta Dinophysis (Dinoflagellates) Chrysophyta Chromulina, Botrydium Phaeophyta (Brown algae) Sargassum, Ectocarpus Cyanophyta (Blue Green Nostoc, Anabaena algae) Rhodophyta (Red algae) Gracillaria, Economic importance of algae  PROTOZOA Protozoa are unicellular mostly microscopic organisms and are classified under subkingdom protozoa of the kingdom protista. Protozoa are eukaryotic cells having a distinct nucleus as well as endoplasmic reticulum, golgi apparatus, mitochondria etc. in the cytoplasm. 'Proto' means first and 'zoan' means animals – first animal life which appear in this universe. The flagellates are the most primitive form of animal life and the ciliates are the most highly organized form of protozoa. The study of these eucaryotic protists is called protozoology.  MORPHOLOGY Size: They have wide variation in size and shape e.g. Leishmania donovani has a size range of 1-4µm. While ciliates range about 2000 µm /2mm. Intracellular structure: Like all eukaryotic cells, protozoan cells consists of cytoplasm separated from the surrounding media by a special cell envelope. Cytoplasm: cytoplasm is differentiated into endoplasm and ectoplasm. The ectoplasm is more gel like and endoplasm is more fluid like. Structures are formal in endoplasm. These include endoplasmic reticulum, Golgi complex, mitochondria, food vacuoles, contractile vacuoles and nuclei. AMOEBA Contractile Vacuole: Contractile vacuole mainly occur in free living aquatic protozoa. They are extremely small rounded floating vacuoles appear in the cytoplasm which coalesce to form a contractile vacuole having osmoreglatory function and maintain the water balance in the body. Food vacuoles are the sites where food particle passes and digestion take place. Food vacuole are having the seats of enzymatic activity. Locomotion: They possess different types of locomotory organs. They may bear flagella (flagellates), cilia (ciliates) or pseudopodia. Locomotory organs are absent in the parasitic forms. Nucleus: The protozoan cells have at least one eukaryotic nucleus however many have multiple nuclei e.g. all ciliates. The protozoan nuclei are of various forms sizes and structures. In several species each individual organism has two similar nuclei in ciliates two dissimilar nuclei. One large macro nucleus and one small micronucleus. Macro nucleus controls metabolic activities and regeneration PARAMECIUM process. The micro nucleus is concerned with reproductive activity. Reproduction: Generally protozoa multiply by asexual reproduction. Many are able to carry out both sexual and asexual reproduction. Some parasitic forms may have an asexual phase in one host and sexual phase in another host e.g. plasmodium. Asexual reproduction: Asexual reproduction occurs by simple cell division by binary fission and multiple fission and budding. Sexual Reproduction: In this fusion of two gametes occur in various groups of protozoa. Conjugation occurs in ciliates.  Control of Microorganisms Physical, Chemical and Biological Methods Physical Methods Chemical Agents Biological Agents Mode of action Conditions Influencing Antimicrobial Activity  Introduction The control of microbial growth is necessary in many practical situations, and significant advances in agriculture, medicine, and food science have been made through study of this area of microbiology. "Control of microbial growth", as used here, means to inhibit or prevent growth of microorganisms. The control of MICROBES in two basic ways: (1) by killing microorganisms or (2) by preventing the growth of microorganisms. Control of growth usually involves the use of physical chemical, and biological agents  Definitions Sterilization: A process that kills all living cells, including viruses and spores, from a substance or object using autoclave Disinfection: A treatment that reduces the total number of microbes on an object or surface, but does not necessarily remove or kill all of the microbes Sanitation: Reduction of the microbial population to levels considered safe by public health standards Antiseptic: A mild disinfectant agent suitable for use on skin surfaces -cidal: A suffix meaning that “the agent kills.” For example, a bacteriocidal agent kills bacteria -static: A suffix that means “the agent inhibits growth.” For example, a fungistatic agent inhibits the growth of fungi, but doesn’t necessarily kill it. Physical Methods Moist Heat Dry Heat Low Temperatures Filtration Radiation Physical Methods: Moist Heat Mechanism of killing is a combination of protein/nucleic acid denaturation and membrane disruption Effectiveness Heavily dependent on type of cells present as well as environmental conditions (type of medium or substrate) Bacterial spores much more difficult to kill than vegetative cells Physical Methods: Moist Heat Measurements of killing by moist heat Thermal death point (TDP): Lowest temperature at which a microbial suspension is killed in 10 minutes; misleading because it implies immediate lethality despite substrate conditions Thermal death time (TDT): Shortest time needed to kill all organisms in a suspension at a specified temperature under specific conditions; misleading because it does not account for the logarithmic nature of the death curve (theoretically not possible to get down to zero) Physical Methods: Moist Heat Measurements of killing by moist heat (cont.) Decimal reduction time (D value): The time required to reduce a population of microbes by 90% (a 10-fold, or one decimal, reduction) at a specified temperature and specified conditions z value: The change in temperature, in ºC, necessary to cause a tenfold change in the D value of an organism under specified conditions F value: The time in minutes at a specific temperature (usually 121.1°C or 250 °F) needed to kill a population of cells or spores Physical Methods: Moist Heat Methods of Moist Heat Boiling at 100°C Effective against most vegetative cells; ineffective against spores; unsuitable for heat sensitive chemicals & many foods Autoclaving/pressure canning Temperatures above 100°C achieved by steam pressure Most procedures use 121.1°C, achieved at approx. 15 psi pressure, with 15 - 30 min autoclave time to ensure sterilization Sterilization in autoclave in biomedical or clinical laboratory must by periodically validated by testing with spores of Clostridium or Bacillus stearothermophilus Physical Methods: Moist Heat Methods of Moist Heat Pasteurization is a process of heating a food, which is usually a liquid, to a specific temperature for a predefined length of time and then immediately cooling it after it is removed from the heat. This process slows spoilage caused by microbial growth in the food. Pasteurization  Used to reduce microbial numbers in milk and other beverages while retaining flavor and food quality of the beverage  Retards spoilage but does not sterilize  Traditional treatment of milk, 63°C for 30 min  Flash pasteurization (high-temperature short term pasteurization); quick heating to about 72°C for 15 sec, then rapid cooling Physical Methods: Moist Heat  Physical Methods: Dry Heat Incineration Burner flames Electric loop incinerators Air incinerators used with fermenters; generally operated at 500°C Oven sterilization Used for dry glassware & heat-resistant metal equipment Typically 2 hr at 160°C is required to kill bacterial spores by dry heat: this does not include the time for the glass to reach the required temp (penetration time) nor does it include the cooling time Physical Methods: Low Temperatures Refrigerator: around 4°C inhibits growth of mesophiles or thermophiles; psychrophiles will grow Freezer: “ordinary” freezer around -10 to -20°C “ultracold” laboratory freezer typically -80°C Generally inhibits all growth; many bacteria and other microbes may survive freezing temperatures  Physical Methods: Filtration Used for physically removing microbes and dust particles from solutions and gasses; often used to sterilize heat-sensitive solutions or to provide a sterilized air flow Depth filters: eg. Diatomaceous earth, unglazed porcelean Membrane filters: eg. Nitrocellulose, nylon, polyvinylidene difluoride HEPA filters: High efficiency particulate air filters used in laminar flow biological safety cabinets Physical Methods: Radiation Ultraviolet Radiation DNA absorbs ultraviolet radiation at 260 nm wavelength This causes damage to DNA in the form of thymine dimer mutations Useful for continuous disinfection of work surfaces, e.g. in biological safety cabinets Physical Methods: Radiation Ionizing Radiation Gamma radiation produced by Cobalt-60 source Powerful sterilizing agent; penetrates and damages both DNA and protein; effective against both vegetative cells and spores Often used for sterilizing disposable plastic labware, e.g. petri dishes; as well as antibiotics, hormones, sutures, and other heat-sensitive materials Also can be used for sterilization of food; has been approved but has not been widely adopted by the food industry Chemical Agents Phenolics Alcohols Halogens Heavy metals Quaternary Ammonium Compounds Aldehydes Peptides Phenolic phenol and phenolics (phenol derivatives) such as cresols, s xylenols, and orthophenylphenol - disinfectants in laboratories and hospitals. Phenolics act by denaturing proteins and disrupting cell membranes. advantages as disinfectants: phenolics are tuberculocidal, effective in the presence of organic material, and remain active on surfaces long after application. Alcohols have a disagreeable odor and can cause skin irritation. most widely used disinfectants and antiseptics. They are bactericidal and fungicidal but not sporicidal; some lipid-containing viruses are also destroyed. The two most popular alcohol germicides are ethanol and isopropanol, usually used in about 70 to 80% concentration. denaturing proteins and possibly by dissolving membrane lipids. A 10 to 15 minute soaking is sufficient to disinfect thermometers and small instruments. Halogen fluorine, chlorine, bromine, iodine, and astatine s The halogens iodine and chlorine are important antimicrobial agents. used as a skin antiseptic and kills by oxidizing cell constituents and iodinating cell proteins. At higher concentrations- even kill some spores. Iodine - tincture of iodine, 2% or more iodine in a water-ethanol solution of potassium iodide. Iodophor-release iodine slowly to minimize skin burns and irritation. Heavy used in hospitals for preoperative skin degerming and disinfecting. Metals mercury, silver, arsenic, zinc, and copper - used as germicides. 1% solution of silver nitrate is often added to the eyes of infants to prevent ophthalmic gonorrhea (in many hospitals, erythromycin is used instead of silver nitrate because it is effective against Chlamydia as well as Neisseria). Silver sulfadiazine is used on burns. Copper sulfate is an effective algicide in lakes and swimming pools. Heavy metals combine with proteins, often with their sulfhydryl groups, and inactivate them. They may also precipitate cell proteins. Quaternary Ammonium Anionic, non-ionic and cationic Compounds cationic detergents are effective disinfectants. The most popular of these disinfectants are quaternary ammonium compounds characterized by a positively charged quaternary nitrogen and a long hydrophobic aliphatic chain They disrupt microbial membranes and may also denature proteins. Eg.: benzalkonium chloride and cetylpyridinium chloride Aldehydes aldehydes, formaldehyde and glutaraldehyde - are highly reactive molecules that combine with nucleic acids and proteins and inactivate them, probably by crosslinking and alkylating molecules They are sporicidal and can be used as chemical sterilants. Glutaraldehyde is less irritating than formaldehyde - used to disinfect hospital and laboratory equipment.  Sterilizing gases Many heat-sensitive items such as plastic Petri dishes, heart- lung machine components, sutures, and catheters are sterilized with ethylene oxide gas. Ethylene oxide (EtO) is both microbicidal and sporicidal. It is a strong alkylating agent that kills by reacting with DNA and proteins to block replication and enzymatic activity. It is a particularly effective sterilizing agent because it rapidly penetrates packing materials, even plastic wraps. Chlorine dioxide (ClO2) gas is also used as a disinfectant. It has been used to sterilize hospital operating and patient rooms. ClO2 fumigation is used in the food industry to sanitize fruits and vegetables of contaminating yeasts and molds. Vaporized hydrogen peroxide (VHP) can also be used to decontaminate biological safety cabinets, operating rooms, and other large facilities. During the course of the decontamination process, VHP breaks down into water and oxygen, both of which are harmless.  Antibiotics Antibiotics are generally considered to be organic compounds of low molecular weight produced by microbes. At low concentration, antibiotics are deleterious to the growth or other metabolic activities of other microorganisms.  Most of the Antibiotics produced by Microorganisms Antibiotic Producing microorganism Cephalosporin Cephalosporium acrimonium Chloramphenicol Streptomyces venezuelae Erythromycin Streptomyces erythreus Griseofulvin Penicillium griseofulvin Penicillin Penicillium chrysogenum Streptomycin Streptomyces griseus Tetracycline Streptomyces aureofaciens Gentamicin Micromonospora purpurea  Penicillin All penicillin like antibiotics inhibit synthesis of peptidoglycan, an essential part of the cell wall. They do not interfere with the synthesis of other intracellular components. These antibiotics do not affect human cells because human cells do not have cell walls. Penicillins are active against Gram positive bacteria Some members (e.g. amoxicillin) are also effective against Gram negative bacteria PRODUCTION OF PENICILLIN Penicillin was the first important commercial product produced by an aerobic, submerged fermentation First antibiotic to have been manufacture in bulk. Used as input material for some semi synthetic antibiotics. It is fermented in a batch culture Thanks to work by Alexander Fleming (1881- 1955), Howard Florey ( 1898-1968) and Ernst Chain (1906-1979), penicillin was first produced on a large scale for human use in 1943. At this time, the development of a pill that could reliably kill  bacteria was a remarkable development and many lives were saved during World War II because this medication was available. A. Fleming E. Chain H. Florey  A tale by A. Fleming In 1928, Sir Alexander Fleming, a Scottish biologist, observed that Penicillium notatum, a common mold, had destroyed staphylococcus bacteria in culture. He published a report on penicillin and its potential uses in the British Journal of Experimental Pathology. Penicillin Mechanism of Antibiotics Summary of antimicrobial agents affecting cell wall synthesis Agents affecting the cell wall b-lactamase inhibitors Clavulanic acid b-lactam antibiotics Other antibiotics Sulbactam Bacitracin Tazobactam Vancomycin Daptomycin Penicillins Cephalosporins Carbapenems Monobactams Amoxicillin Ertapenem Imipenem/cilastatin* Aztreonam Ampicillin Meropenem Dicloxacillin Indanyl carbenicillin Methicillin 1st generation 2nd generation 3rd generation 4th generation Nafcillin Cefadroxil Oxacillin Cefaclor Cefdinir Cefepime Cefazolin Cefprozil Cefixime Penicillin G Cephalexin Cefuroxime Penicillin V Cefotaxime Cefoxitin Ceftazidime Piperacillin Ceftibuten Ticarcillin Ceftizoxime Ceftriaxone (according to Lippincott´s Pharmacology, 2009)  Peptidoglycan Synthesis “Penicillin binding protein” P. aeruginosa treated with Rifampin propidium iodide ------ viable cells counting ethidium bromide ----- dead cells counting Control (without Anti) 5 µg/mL, 30 min 25 µg/mL, 30 min Summary of antimicrobial agents affecting cell wall synthesis Agents affecting the cell wall b-lactamase inhibitors b-lactam antibiotics Other antibiotics Clavulanic acid Sulbactam Bacitracin Tazobactam Vancomycin Daptomycin Penicillins Cephalosporins Carbapenems Monobactams Amoxicillin Ertapenem Imipenem/cilastatin* Aztreonam Ampicillin Meropenem Dicloxacillin Indanyl carbenicillin 1st generation 2nd generation 3rd generation 4th generation Methicillin Nafcillin Cefadroxil Cefaclor Cefepime Oxacillin Cefdinir Cefazolin Cefprozil Cefixime Penicillin G Cephalexin Cefuroxime Cefotaxime Penicillin V Cefoxitin Ceftazidime Piperacillin Ceftibuten Ticarcillin Ceftizoxime Ceftriaxone The Beta-Lactam Antibiotics Cell wall active agents Prevent the final step in the synthesis of the bacterial cell wall Range from very narrow spectrum to very broad spectrum β-lactam ring How do they work? 1. The β-lactam binds to Penicillin Binding Protein (PBP) 2. PBP is unable to crosslink peptidoglycan chains 3. The bacteria is unable to synthesize a stable cell wall 4. The bacteria is lysed (cidal effects). bacteriacidal effect against G+&G- Penicillin-β lactum antibiotic Cephalosporin The cephalosporins are a class of β-lactam antibiotics originally derived from the fungus Acremonium, which was previously known as "Cephalosporium". Cephamycins constitute a subgroup of β-lactam antibiotics called cephems. A lactam is a cyclic amide. The term is a lactone + amide. β-lactam 108 Mode of action - Cephalosporins Cephalosporins are bactericidal and have the same mode of action as other β-lactam antibiotics (such as penicillins), but are less susceptible to β- lactamases. Cephalosporins disrupt the synthesis of the peptidoglycan layer of bacterial cell walls. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidases known as penicillin-binding proteins (PBPs). PBPs bind to the D-Ala-D-Ala at the end of muropeptides (peptidoglycan precursors) to crosslink the peptidoglycan. Active against both Gram positive and negative bacteria  Peptidoglycan Synthesis “Penicillin binding protein” Conditions Influencing Antimicrobial Activity Several critical factors play key roles in determining the effectiveness of an antimicrobial agent, including: Population size Types of organisms Concentration of the antimicrobial agent Duration of exposure Temperature pH Organic matter Broad spectrum antibiotics- Antibiotics from procaryotes  Most of the Antibiotics produced by Microorganisms Antibiotic Producing microorganism Cephalosporin Cephalosporium acrimonium Chloramphenicol Streptomyces venezuelae Erythromycin Streptomyces erythreus Griseofulvin Penicillium griseofulvin Penicillin Penicillium chrysogenum Streptomycin Streptomyces griseus Tetracycline Streptomyces aureofaciens Gentamicin Micromonospora purpurea  ANTIBIOTICS  Classification according chemical structure similarity  I. ß-Lactam antibiotcs  II. The Aminoglycoside Antibiotics  III. The Macrolide Antibiotics  IV : Antibiotics with fused ring systems:  V. Lincomycins  VII.Polypeptide Antibiotics  VIII.Unclassified antibiotics A. Penicillin Natural penicillin Penicillin G; benzylpenicillin ß-Lactam thiazolidine penam penicillin penicillanic acid  Ampicillin 6-[D-a-Aminophenylacetamido] penicillanic acid; 6--amino-benzyl penicillin Amoxicillin, 6-[D-(-)--Amino-p-hydroxy-phenylacetamido] penicillanic acid (Amoxil). The incorporation of an acidic substiuent at the α-benzyl carbon atom of penicillin G also imparts clinical effectiveness against Gram- negative bacilli and, furthermore, extends the spectrum of activity to include organisms that are resistant to ampicillin. Mode of action of penicillin Inhibit cell wall synthesis by acylation of transpeptidase enzyme necessary for synthesis of dipeptidogycan which responsible for rigidity and strength of the cell wall  ß-Lactamase inhibitors Clavulanate Potassium: Clavulanic acid is an antibiotic isolated from Streptomyces clavuligeris. Structurally it is 1-oxapenam lacking the 6-acylamino side chain of penicillin, but possessing a 2-hydroxyethylidene moiety at C-2. Sulbactam This synthetic penicillin derivative is a potent inhibitor of ß-lactamase. It potentiates the activity of ampicillin and carbenicillin against ß -lactamase producing bacteria. Combinations of amoxicillin and the potassium salt of clavulanic acid are available (Augmentin) in a variety of fixed-dose, oral dosage forms intended for the treatment of skin, respiratory, ear and urinary tract infections caused by ß-lactamase producing bacterial strains resistant to amoxicillin alone. 119  B- Cephalosporins: Cephalosporins are ß-lactam antibiotics isolated from Cephalosporium species or prepared semisynthetically Semisynthetic Derivatives: In the preparation of semisynthetic cephalosporins, the following improvements are sought: (1) increased acid stability; (2) improved pharmacokinetic properties, particularly better oral absorption; (3) broadened antimicrobial spectrum; (4) increased activity against resistant microorganisms (5) decreased allergenicity; and (6) increased tolerance after parenteral administration Cephradine, USP (Velosef) Available oral and parentral Cefadroxil, USP (Duricef ) Slowly excreted , longer duration HO of action O H H S N H H2 O NH 2 N O CH 3 COOH Cafaclor USP (Ceclor) More potent against Hemophiles influenza  II. Aminoglycoside Antibiotics Aminologlycosides are so named because their structures consist of amino sugars linked glycosidically. The streptomycin, neomycin, paromomycins, gentamicins, Tobramycins, Kanamycins, and Amikacins have many chemical and antimicrobial features in common: All these antibiotics show broad spectrum antimicrobial activity Paromomycin inhibits Enatmoeba histolytica. neomycin - used widely in the treatment of intestinal infections and chemosterilization of the bowel prior to surgery of that organ Mechanism of Action The amino glycosides - act directly on the bacterial ribosome to inhibit the initiation of protein synthesis - and to interfere with the fidelity of translation of the genetic message. They bind to the 30S ribosomal subunit to form a complex that is unable to initiate proper amino acid polymerization  III. Macrolide Antibiotics Macrolides are a group of macrocyclic antibiotics containing: An amino sugar linked glycosidically to the lactone ring, A neutral sugar linked to the ring or the basic sugar and contains a ketone group. The macrolides are principally active against Gram positive bacteria and show useful activity against penicillin-resistant strains. Also exhibit effectiveness against gram-negative cocci. Mode of action: Bacteriostatic, bind to 50S ribosomal subunit to prevent the translocation step of bacterial protein synthesis 124 Eg: Erythrommycin, Azithromycin  IV : Antibiotics with fused ring systems: The group includes the broad-spectrum tetracycline. The Tetracycline Comprises a group of antibiotics characterized by their common octahydronaphthacene skeleton. Other names R4 R3 R2 R1 Name Achromycin H CH3 OH H a. Tetracycline oxycycline Cl CH3 OH H b. 7-Chlortetracyclin Terramycin H CH3 OH OH c. 5-Oxytetracyclin Demeclocycl Cl H OH H d.6-Demethyl-7-chloro tetracyclin Methacycline H = CH2 OH e 6-Demethy1-6-deoxy-5-hydroxy-6- Doxycycline methylene tetracycline Minocycline H CH3 H OH f. 6-Deoxy-5-oxytetracycline N(CH3)2 H H H g. 7-Dimethylamino 6-demethyl-6- deoxytetracycline V. Lincomycins They are known as Sulphur containing Antibiotics Mode of action: act via 50S ribosomal subunit binding & protein synthesis inhibition. They are used in extra CNS anaerobic infections, Penicillin sensitive patients CLINDAMYCIN LINCOMYCIN 7S-Cloro-7S-deoxylincomycin semisynthetic  VI.Polypeptide Antibiotics The most powerful antibiotic agents but limited for renal toxicity. Used mainly locally in burns. Mode of action: Inhibit mucopeptide cell wall synthesis and interfere with semipermeability of cell membrane BACITRACIN GRAMICIDIN Val–Orn–Leu– D -Phe–Pro Pro– D -Phe–Leu–Orn–Val POLYMYXIN (B) 127 VII. Unclassified Antibiotics CHLORAMPHENICOL In meningitis, typhoid & paratyphoid fever. D-(-) threo-2-Dichloroacetamido --1-(4– nitrophenyl)-1,3-propanediol Thiamphenicol CH3SO2-  Antimicrobial Drug Resistance Is a Public Health Threat The spread of antibiotic-resistant bacteria has become so widespread that methicillin- resistant S. aureus (MRSA) has become well known. Fortunately, most MRSA strains can still be treated, but the more recent emergence of vancomycin-resistant enterococci (VRE) and carbapenem-resistant Enterobacteriaceae (CRE) bacteria has caused great concern among public health officials worldwide. There are two types of resistance: intrinsic and acquired. An example of intrinsic resistance is that of mycoplasma resistance to β-lactam antibiotics and other cell wall inhibitors simply because these bacteria lack a cell wall. Acquired resistance occurs when there is a change in the genome of a bacterium that converts it from one that is sensitive to an antibiotic to one that is resistant.  MECHANISMS FOR ACQUIRING RESISTANCE Bacteria use several mechanisms to become antibiotic-resistant: Inactivation of the antibiotic Efflux pumping of the antibiotic Modification of the antibiotic target Alteration of the pathway INACTIVATION OF ANTIBIOTIC Inactivation involves enzymatic breakdown of antibiotic molecules. A good example is β-lactamase: Secreted into the bacterial periplasmic space Attacks the antibiotic as it approaches its target There are more than 190 forms of β-lactamase. E.g of lactamase activity in E.coli and S. aureus MECHANISMS FOR ACQUIRING RESISTANCE Destruction of normal flora allows pathogenic pathogens to dominate  MRSA, VRSA, VRE, AND OTHER PATHOGENS  Several antibiotic-resistant bacteria are considered clinically dangerous. › MRSA (Methicillin-Resistant) and VRSA (Vancomycin- resitant S.aureus are very virulent in humans and are referred as professional pathogens.  MRSA and VRSA contain many resistance genes. › Three or four resistance islands on the chromosome › 26-28 additional gene clusters on plasmids which can move to other bacterial cells. › VRE-Vancomycin enterococcus e.g E. faecalis contributes to 90% of all vancomycin resistant bacteria Clostridium difficile is a superinfection pathogen. Establishes itself in the intestinal tract as part of a superinfection It is very resistant to antibiotics. Patients with this infection are difficult to treat Guidelines for extending the useful life of antimicrobial drugs Antifungal agents  Antifungal agents Human fungal infections have increased dramatically in recent years Fungal infections are usually more difficult to treat than bacterial infections, because fungal organisms grow slowly - because fungal infections often occur in tissues that are poorly penetrated by antimicrobial agents Therapy of fungal infections usually requires prolonged treatment. They are complex organisms in comparison to bacteria.Thus antibacterial agents are not effective against fungi. Fungal infections are also called as mycoses  They have nucleus and well defined nuclear membrane, and chromosomes. they have rigid cell wall composed of chitin (N – acetylglucosamine ) where as bacterial cell wall is composed of peptidoglycan fungal cell membrane contains ergosterol , human cell membrane is composed of cholesterol Fungal infections SUPERFECIAL Dermatomycoses affecting skin, hair or nails. Epidermophyton (skin and nails) Trichophyton (skin,hair & nail) Microsporum (skin and hair) b) Candidiasis (commonly normal flora of mouth, skin, intestines and vagina) infection caused by genus Candida affecting skin, mucous membrane of mouth or G.I.T or female genital tract SYSTEMIC Candidiasis ,Cryptococosis, Aspergillosis, Blastomycosis, Histoplasmosis, Coccidioidomycosis, Paracoccidioidomycosis etc. Drug Classification A) Drugs that disrupt fungal cell membrane i) Polyenes b) Triazole Fluconazole Itraconazole Tioconazole Amphotericin Nystatin iii) Allylamines Terbinafine Natamycin ii) Azoles Naftifine a) Imidazole Butenafine iv) Echinocandins Ketoconazole caspofungin, Butaxonazole Clotrimazole Econazole Miconazole Oxiconazole Sulconazole Drug classification cot’d B) Drugs that inhibits mitosis Griseofulvin C) Drugs that inhibits DNA synthesis flucytosine D) Miscellaneous Haloprogi Tolnaftate Whitefield's ointment Ciclopirox olamine Mechanism of action of different anti fungal drugs  Uses of antifungal drugs Drug used Disease Systemic infections Amphotericin, flucytocin, , fluconazole. systemic candidiasis Amphotericin, flucytocin , fluconazole, Cryptococcosis( meningitis) itraconazole itraconazole Amphotericin, systemic aspergillosis itraconazole Amphotericin, Blastomycosis Amphotericin, itraconazole ,fluconazole. Histoplasmosis fluconazole. itraconazole ,Amphotericin, Coccidiomycosis fluconazole. itraconazole ,Amphotericin, Paracoccidiomycosis Amphotericin, flucytocin ,Amphotericin, Mucormycosis Amphotericin, flucytocin Disseminated sportrichosis Antiviral Drugs 145  Antiviral Drugs Vaccines are often used to build up immunity before a viral infection occurs. Common viral infections such as the influenza, mumps, or chicken pox are usually overcome by the body’s immune system. To be effective, antiviral agents must either block viral entry into or exit from the cell or be active inside the host cell. 146  Antiviral drugs work by: 1.Altering the cell’s genetic material so that the virus cannot use it to multiply, i.e. acyclovir (inhibiting Viral enzymes, Host expression of viral proteins & Assembly of viral proteins) 2.Preventing new virus formed from leaving the cell, i.e. amatadine.  Antiviral therapy challenging. 1. Rapid replication of viruses makes it difficult to develop effective antiviral. 2. Viruses can rapidly mutate and drug becomes ineffective. 3. Difficulty for drug to find virus without injuring normal cells.(Nonselective inhibitors of virus replication may interfere with host cell function and result in toxicity.) Antiviral drugs share the common property of being virustatic; they are active only against replicating viruses and do not affect latent virus. 148  Agents To Treat Herpes Simplex Virus (HSV) & Varicella-zoster Virus (VZV) Infections Oral Agents Topical Agents Acyclovir Acyclovir Valacyclovir Docosanol Famciclovir Penciclovir Ophthalmic Intravenous Trifluridine Acyclovir  Herpes simplex viruses (HSV)—cause repeated, blister-like lesions on the skin, genitals, mucosal surfaces. Some remain latent; activated by physical or emotional stress HSV-type 1—non genital HSV type 2—genital infections Acyclovir Valacyclovir is a Acyclovir prodrug, with better Herpes virus availability specific thymidine kinase Acyclovir is Guanosine analog Acyclovir monophosphate mostly taken up by the virus infected cells and Host kinase has low toxicity for host Acyclovir cells. triphosphate 1. Incorporated into DNA and terminates synthesis 2. Inhibition of herpes virus DNA polymerase Acyclovir. Clinical Use Herpes simplex Herpes zoster Chickenpox Epstain-Barr virus  Ganciclovir Valganciclovir ( a prodrug) Mechanism like Acyclovir Active against all Herpes viruses & CMV Low oral bioavailability given I.V. Most common A/E: bone marrow suppression (leukopenia, thrombocytopenia ) and CNS effects (headache, psychosis, convulsions). 1/3 of patients have to stop because of adverse effects  Foscarnet An inorganic pyrophosphate analog does not have to be phosphorylated Active against Herpes (I, II, Varicella , CMV), including those resistant to Acyclovir and Ganciclovir. IV only MOA:Direct inhibition of DNA polymerase and RT Foscarnet should only be given during pregnancy when benefit outweighs risk. Cidofovir Incorporation into viral DNA chain results in reductions of the rate of viral DNA synthesis Must be administered with high-dose probenecid & adequate hydration Antiretroviral Agents  Retroviruses are enveloped, single stranded RNA viruses that replicate through a DNA intermediate using Reverse Transcriptase. This enzyme converts the RNA genome into DNA, which then integrates into the host chromosomal DNA by the enzyme Integrase. This large and diverse family includes members that are oncogenic, are associated with a variety of immune system disorders, and cause degenerative and neurological syndromes. Current classes of antiretroviral drugs include: Three main enzymatic targets:  Reverse Transcriptase  Protease  Integrase six drug classes 1. Nucleoside Reverse Transcriptase Inhibitors (NRTIs) 2. Non Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) 3. Protease inhibitors (PIs) 4. Entery inhibitors 5. CCR5 receptor antagonists 6. Integrase inhibitors Lantibiotics  Lantibiotics spread of bacterial resistance leads to a growing demand for novel antibiotics Bacteriocins Amino acid sequence and bridging pattern of selected representatives of type A and type B lantibiotics. Type A lantibiotics kill bacteria by forming pores in the cytoplasmic membrane type B lantibiotics depicted inhibit cell wall biosynthesis. Nisin is an antimicrobial peptide produced by certain Lactococcus species. Nisin has been accepted as a safe and natural preservative for milk and other food products in more than 50 countries This peptide inhibits the vegetative growth of a range of gram-positive bacteria nisin inhibits the food-borne pathogens Listeria monocytogenes, Staphylococcus aureus, and psychrotrophic enterotoxigenic Bacillus cereus, the effectiveness of nisin as a food preservative against these organisms under various preservation conditions Bacteriocins are toxins produced by bacteria directed at killing a limited number of close relatives.