Microbial Diversity By Dr. B. KAYATHRI, ASSISTANT PROFESSOR, DEPARTMENT OF BIOTECHNOLOGY

1. Microbial Diversity By Dr. B. KAYATHRI,ASSISTANT PROFESSOR,DEPARTMENT OF BIOTECHNOLOGY

2. Categories of Microbes • Microbes can be divided into those that are truly cellular (bacteria, archaea, algae, protozoa, and fungi) and those that are acellular (viruses, viroids, and prions). • Cellular microbes (microorganisms) can be divided into those that are prokaryotic (bacteria and archaea) and those that are eukaryotic (algae, protozoa, and fungi). • Viruses, viroids, and prions are often referred to as acellular microbes or infectious particles.

3. Acelluar Microbes: Viruses • Complete virus particles are called virions. • Very small and simples in structure (10 – 300 nm) • No type of organism is safe from viral infection • Some viruses, called oncogenic viruses or oncoviruses, cause specific types of cancer. • A typical virion consists of a genome of either DNA or RNA, surrounded by a capsid (protein coat), which is composed of protein units called capsomeres. • Some viruses (enveloped viruses) have an outer envelope composed of lipids and polysaccharides.

4. Viral Nucleocapsids and Envelopes • v

5. Acellular Microbes: Viruses • Viruses have five properties that distinguish them from living cells: • They possess either DNA or RNA, whereas living cells possess both. • They are unable to replicate on their own. • Unlike cells, they do not divide by binary fission, mitosis, or meiosis. • They lack the genes and enzymes necessary for energy production. • They depend on the ribosomes, enzymes, and metabolites of the host cell for protein and nucleic acid production.

6. Viruses that Infect Humans (Fig. 4-1)

7. Acellular Microbes: Viruses • Viruses are classified by • Type of genetic material (either DNA or RNA) • Shape and size of capsid • Number of capsomeres • Presence or absence of an envelope • Type of host it infects • Disease it produces • Target cell(s) • Immunologic/antigenic properties

9. Acellular Microbes: Viruses • Categories based on genome • Double-stranded DNA viruses • Single-stranded RNA viruses • Single-stranded DNA viruses • Double-stranded RNA viruses • Most viral genomes are circular molecules, but some are linear.

10. Acellular Microbes: Viruese • Origin of Viruses • Coevolution theory - coevolved with bacteria and archaea from the primordial soup • Retrograde evolution theory – viruses evolved from free-living prokaryotes that invaded other living organisms, and gradually lost functions which were provided by the host cell • Escaped gene theory – viruses are a piece of host cell RNA or DNA that have escaped from living cells and are no longer under cell control -- currently most widely accepted

11. Acellular Microbes: Viruses • Bacteriophages • Viruses that infect bacteria are known as bacteriophages or simply phages. • Categorized based on shape, nucleic acid, and events that occur after entry into a host cell • Shape: icosahedron, filamentous, complex • Nucleic acid: ss DNA, ds DNA, ss RNA, ds RNA • Events: virulent vs temperate

12. Acellular Microbes: Viruses • Virulent bacteriophages vs. temperate bacteriophages. • Virulent bacteriophages always cause what is known as the lytic cycle, which ends with the destruction of the bacterial cell. • The five steps in the lytic cycle are attachment, penetration, biosynthesis, assembly, and release.

13. Acellular Microbes: Viruses • Lytic cycle of bacteriophages

14. Acellular Microbes: Viruses • Temperate bacteriosphages (lysogenic phages) • Do not immediately initiate the lytic cycle • Instead, integrates their DNA into the bacterial cell chromosome • Phage switches to lytic cycle as a result of environmental clues

15. Acellular Microbes: Viruses • Uses of bacteriophages • Destroy bacterial pathogens • Treat bacterial infection • Prevent food contamination

16. Acellular Microbes: Viruses • Animal Viruses • Infect animals or humans • DNA or RNA • Simple or complex • Steps in the multiplication of animal viruses are • Attachment • Penetration • Uncoating • Biosynthesis • Assembly • Release

17. Acellular Microbes: Viruses • Latent virus infections • Viral infections in which the virus is able to hide from a host’s immune system by entering cells and remaining dormant. • Herpes viral infections are examples. • Once acquired, herpes virus infections (e.g., those that cause cold sores, genital herpes, and chickenpox/shingles) never completely go away; for example, chickenpox may be followed, years later, by shinglesboth the result of the same virus.

18. Acellular Microbes: Viruses • Antiviral agents • Antibiotics are not effective against viral infections • Antiviral agents are drugs that are used to treat viral infections. • These agents interfere with virus-specific enzymes and virus production by disrupting critical phases in viral multiplication or inhibiting synthesis of viral DNA, RNA, or proteins.

19. Acellular Microbes: Viruses • Oncogenic viruses or oncoviruses • These viruses cause cancer. • Examples include Epstein–Barr virus, human papillomaviruses, Hepatitis B and C, and human herpesvirus 8 • Human immunodeficiency virus (HIV) • This virus causes acquired immunodeficiency syndrome (AIDS). • It is an enveloped, single-stranded RNA retrovirus. • The primary targets for HIV are CD4+ cellsthose having CD4 receptors on their surface.

20. Acellular Microbes: Viruses • Plant Viruses • Cause plant disease • Result in economic losses in excess of $70 billion per year worldwide • Infects cocoa tree, rice, barley, tobacco, turnips, cauliflower, potatoes, tomatoes, etc.

21. Acellular Microbes: Viroids and Prions • Viroids and prions are smaller and less complex infectious particles than viruses • Viroids • Viroids are short, naked fragments of single-stranded RNA, which can interfere with the metabolism of plant cells. • Viroids are transmitted between plants in the same manner as viruses. • Examples of plant diseases caused by viroids are potato spindle tuber and citrus exocortis. • Does not cause animal disease

22. Acellular Microbes: Viroids and Prions • Prions • Prions are small infectious proteins that cause fatal neurologic diseases in animals and humans • Scrapie • mad cow disease • Kuru • Creutzfeldt–Jacob disease • Fatal Familial insomnia • Of all pathogens, prions are the most resistant to disinfectants. • The mechanism by which prions cause disease remains a mystery. All are fatal spongiform encephalopathies

23. Domain Bacteria: Characteristics • Bergey’s Manual of Systematic Bacteriology • Bacteriologist’s “bible” • Domain bacteria contains • 23 phyla • 32 classes • 5 subclasses • 77 orders • This may only account for <1% to a few percent of the total number of bacteria present in nature • 14 suborders • 182 families • 871 genera • 5,007 species

24. Domain Bacteria: Characteristics • Bacteria are divided into three major phenotypic categories: • Those that are Gram-negative and have a cell wall • Those that are Gram-positive and have a cell wall • Those that lack a cell wall (Mycoplasma spp.) • Characteristics of bacteria used in classification and identification include cell morphology, staining reactions, motility, colony morphology, atmospheric requirements, nutritional requirements, biochemical and metabolic activities, enzymes that the organism produces, pathogenicity, and genetic composition.

25. Domain Bacteria: Cell Morphology • There are three basic categories of bacteria based on shape: • Cocci (round bacteria) • Bacilli (rod-shaped bacteria) • Curved and spiral-shaped bacteria

26. Domain Bacteria: Cell Morphology • Cocci may be seen singly or in pairs (diplococci), chains (streptococci), clusters (staphylococci), packets of 4 (tetrads), or packets of 8 (octads). • Medically relevant: Enterococcus, Neisseria, Staphylococcus, Streptococcus

27. Domain Bacteria: Cell Morphology • Bacilli • They are often referred to as rods; they may be short or long, thick or thin, and pointed or with curved or blunt ends. • They may occur singly, in pairs (diplobacilli), in chains (streptobacilli), in long filaments, or branched. • Extremely short bacilli are called coccobacilli. • Examples of medically important bacilli: Escherichia, Klebsiella, Proteus, Pseudomonas, Haemophilus, and Bacillus spp.

28. Domain Bacteria: Cell Morphology • Curve (comma shaped) • Medically relevant example: Vibrio • usually occur single, but some may form pairs • Spiral shape – called spirochetes • Vary in length, rigidity, size, number and amplitude of their coils, and tightness of coils • Preponema pallidum (syphilis), Borrelia (Lyme disease) • Shape can be lost due to adverse growitn conditions • Some bacteria can exist in a variety of shapes (pleomorphic)

29. Domain Bacteria: Staining Procedures • Most bacteria are colorless, transparent, and difficult to see • Three major categories of staining procedures • Simple staining procedure • Structural staining procedures • Capsule staining • Spore staining • Flagella staining • Differential staining procedures • Gram and acid-fast staining procedures

30. Domain Bacteria: Staining Procedures • Bacterial smears must be fixed prior to staining. • The fixation process serves to kill organisms, preserve their morphology, and anchor the smear to the slide. • The two most common techniques of fixation: • Heat fixationnot a standardized technique; excess heat will distort bacterial morphology • Methanol fixationa standardized technique; the preferred method

31. Simple Bacterial Staining Technique

32. Domain Bacteria: Staining Procedures • Gram Stain • Divides bacteria into two major groups: • Gram-positive (bacteria end up being blue to purple) • Gram-negative (bacteria end up being pink to red) • The final Gram reaction (positive or negative) depends on the organism’s cell wall structure. • The cell walls of Gram-positive bacteria have a thick layer of peptidoglycan, making it difficult to remove the crystal violet–iodine complex. • Gram-negative organisms have a thin layer of peptidoglycan, making it easier to remove the crystal violet; the cells are subsequently stained with safranin.

33. Gram stain Technique

34. Domain Bacteria: Staining Procedures • Some bacteria are neither consistently purple nor pink after Gram staining; they are known as Gram-variable bacteria, for example, Mycobacterium spp. • Mycobacterium spp. are often identified using the acid-fast stain. • The acid-fast stain • Carbol fuchsin is the red dye that is driven through the bacterial cell wall using heat. • Heat is used to soften the waxes in the cell wall. • Because mycobacteria are not decolorized by the acid–alcohol mixture, they are said to be acid-fast.

35. Domain Bacteria: Motility • If a bacterium is able to “swim,” it is said to be motile. • Bacterial motility is most often associated with flagella and less often with axial filaments. • Most spiral-shaped bacteria and about 50% of bacilli are motile; cocci are generally nonmotile. • Motility can be demonstrated by stabbing the bacteria into a tube of semisolid medium

36. Domain Bacteria: Colony Morphology • A bacterial colony contains millions of organisms. • Colony morphology (appearance of the colony) varies from one species to another. • Colony morphology includes size, color, overall shape, elevation, and the appearance of the edge or margin of the colony. • Colony morphology can also include the results of enzymatic activity on various types of media. • As is true for cell morphology and staining characteristics, colony morphology is an important “clue” to the identification (speciation) of bacteria.

37. Domain Bacteria: Atmospheric Requirements • Bacteria can be classified on the basis of their atmospheric requirements, including their relationship to O2 and CO2. • With respect to O2, bacterial isolates can be classified as • Obligate aerobes • Microaerophilic aerobes • Facultative anaerobes • Aerotolerant anaerobes • Obligate anaerobes • Capnophilic organisms grow best in the presence of increased concentrations of CO2 (usually 5%–10%)

38. Domain Bacteria: Atmospheric Requirements

39. Domain Bacteria: Atmospheric Requirements

40. Domain Bacteria: Nutritional Requirements • All bacteria require some form of the elements such as carbon, hydrogen, oxygen, sulfur, phosphorus, and nitrogen for growth. • Some bacteria require special elements (e.g., calcium, iron, or zinc) or vitamins • Organisms with especially demanding nutritional requirements are said to be fastidious (“fussy”). • The nutritional needs of a particular organism are usually characteristic for that species and are sometimes important clues to its identity.

41. Domain Bacteria: Biochemical and Metabolic Activities • As bacteria grow, they produce many waste products and secretions • Pathogenic strains of many bacteria, such as staphylococci and streptococci, can be tentatively identified by the enzymes they secrete. • In particular environments, some bacteria produce gases such as carbon dioxide or hydrogen sulfide. • To identify bacteria in the laboratory, they are inoculated into various substrates (i.e., carbohydrates and amino acids) to determine whether they possess the enzymes necessary to break down those substrates.

42. Domain Bacteria: Pathogenicity • Many pathogens are able to cause disease because they possess capsules, pili, or endotoxins, or because they secrete exotoxins and exoenzymes that damage cells and tissues. • Frequently, pathogenicity is tested by injecting the organism into mice or cell cultures. • Examples of some common pathogenic bacteria: • Neisseria meningitidis, Salmonella typhi, Shigellaspp., Vibrio cholerae, Yersinapestis, and Treponemapallidum

43. Domain Bacteria: Genetic Composition • Laboratory identification of bacteria is moving toward analyzing the organism’s DNA or RNA • The composition of the genetic material (DNA) of an organism is unique to each species. • Through the use of 16S rRNA sequencing, the degree of relatedness between two different bacteria can be determined.

44. Domain Bacteria: Unique Bacteria • Rickettsias, chlamydias, and mycoplasmas are bacteria, but they do not possess all the attributes of typical bacterial cells. • Rickettsias and chlamydias have a Gram-negative type of cell wall and are obligate intracellular pathogens • Rickettsias • Disease transmitted by arthropods • Cause typhus and spotted fever rickettsiosis • have “leaky membranes.”

45. Domain Bacteria: Unique Bacteria • Chlamydias • are “energy parasites,” meaning they prefer to use ATP molecules produced by their host cell. • Transferred by aerosols or direct contact • Cause trachoma, inclusion conjunctivitis, nongonococcal urethritis, pneumonia, and respiratory disease

46. Domain Bacteria: Unique Bacteria • Mycoplasmas • smallest of the cellular microbes. • lack a cell wall and therefore assume many shapes (i.e., pleomorphic) • May be free living or parasitic • Pathogenic to many animals and some plants • In humans, pathogenic mycoplasmas cause primary atypical pneumonia and genitourinary infections. • Because they have no cell wall, they are resistant to drugs like penicillin that attack cell walls.

47. Domain Bacteria: Photosynthetic bacteria • Photosynthetic bacteria include purple bacteria, green bacteria, and cyanobacteria • they all use light as an energy source (photosynthesis), but not in the same way. • Purple and green bacteria do not produce oxygen (anoxygenic photosynthesis), whereas cyanobacteria do (oxygenic photosynthesis) • Cyanobacteria • Believed to be the first organisms capable of carrying out oxygenic photosynthesis • When appropriate conditions exist in a pond or lake, a water bloom will occur • Perform nitrogen fixation • Produce toxins

48. Domain Archaea • Archaea (meaning “ancient”) • they are prokaryotic organisms. • Genetically, archaea are more closely related to eukaryotes than they are to bacteria. • Archaea vary widely in shape; some live in extreme environments, such as extremely acidic, extremely hot, or extremely salty environments. • Archaea possess cell walls, but their cell walls do not contain peptidoglycan (in contrast, all bacterial cell walls contain peptidoglycan)