General charecteristics of bacteria

Dr. Archana S. Nair

1. College Of Veterinary and Animal Sciences Mannuthy Bacteria:- General Characteristics, Pathogenicity, Virulence Factor And Its Immune Response Archana S. N air Department Of Veterinary Public Health

2. Bacterial Introduction Bacteria are unicellular micro-organisms ranging in length from a few micrometers to half a millimeter Bacteria can be found in almost every ecosystem on Earth

3. Bacteria are found 2 billion years before eukaryotes Some bacteria are pathogenic and cause disease

4. Bacterial History Bacteria were first observed by Anton Van Leeuwenhoek in 1676 The term ‘bacteria’, Greek for ‘small stuff’ was first used in 1838

5. Bacterial history Robert Koch and Louis Pasteur were the first to discover that bacteria caused many diseases (mid 19 th century) The first antibiotic used to treat bacterial disease was made by Paul Ehrlich in 1910. It was used to treat Syphilis

6. The Evolution of Bacteria Bacteria are thought (by some) to be the first forms of life, about 4 billion years ago It is believed that both the Domain Archaea and the Domain Eukarya evolved from bacteria

7. Classification

8. Morphology Bacterial cells are prokaryotic, lacking a nucleus and complex organelles They have a cell membrane and a cell wall made up of peptidoglycan

9. Teichoic acid in gram positive bacteria

10. Cell wall

11. Gram negative bacteria

12. Cell membrane Site of biosynthesis of DNA, cell wall polymers and membrane lipids. Selective permeability and transport of solutes into cells Electron transport and oxidative phosphorylation Excretion of hydrolytic exoenzymes

13. Cytoplasm Nucleiod Chromosomal DNA Plasmids Inclusion bodies Storage of excess food and energy Metachromatic granules / Babes ernst granules Much granule Spores Resist adverse condition Ribosomes

14. Bacteria use flagella or pili for movement and interaction with the environment Pili Common pili - fimbriae Sex pili - conjugation

15. Capsule and slime layers Attachment Protection from phagocytic engulfment Resistance to drying Depot for waste products Reservoir for certain nutrients Protection

16. Flagella Some bacterial species are mobile and possess locomotory organelles - flagella. Flagella consist of a number of proteins including flagellin

17. Plasmid Plasmids are small circular ， extrachromosomal , double-stranded DNA molecules They are capable of self-replication and contain genes that confer some properties such as antibiotic resistance, virulence factors

18. Nucleus Lacking nuclear membrane, absence of nucleoli, hence known as nucleic material or nucleoid , one to several per bacterium

19. Inclusions of Bacteria Inclusions are aggregates of various compounds that are normally involved in storing energy reserves or building blocks for the cell

20. Endospores Resistant structure Heat, irradiation, cold Boiling >1 hr still viable Takes time and energy to make spores Location important in classification Central, Subterminal , Terminal Bacillus stearothermophilus -spores Used for quality control of heat sterilization equipment Bacillus anthracis - spores Used in biological warfare

21. Morphological classification Bacteria can be classified into five major groups on morphological basis 1. TRUE BACTERIA Cocci – These are spherical or oval cells. On the basis of arrangement of individual organisms they can be described as Monococci ( Cocci in singles) – Monococcus spp. Diplococci ( Cocci in pairs) – Streptococcus pneumoniae Staphylococci ( Cocci in grape-like clusters) – Staphylococcus aureus Streptococci ( Cocci in chains) – Streptococcus pyogenes Tetrad ( Cocci in group of four) - Micrococcus spp. Sarcina ( Cocci in group of eight)

22. Bacilli – These are rod-shaped bacteria. On the basis of arrangement of organisms, they can be described as Diplobacilli Streptobacilli Palisades Chinese-letter form Coccobacilli Comma-shaped

23. 2. ACTINOMYCETES ( actin - ray, mykes -fungus) These are rigid organisms like true bacteria but they resemble fungi in that they exhibit branching and tend to form filaments

24. 3. Spirochaetes These are relatively longer, slender, non-branched microorganisms of spiral shape having several coils

25. 4. Mycoplasmas These bacteria lack in rigid cell wall (cell wall lacking) and are highly pleomorphic and of indefinite shape They occur in round or oval bodies and in interlacing filaments

26. 5. Rickettsiae and Chlamydiae These are very small, obligate parasites, and at one time were considered closely related to the viruses. Now, these are regarded as bacteria

27. Based on Cultural characteristics Extra growth factors requirements Fastidious – Hemophilus influenzae Non-fastidious – Escherichia coli Hemolysis on Sheep Blood Agar Alpha- hemolysis – Streptococcus pneumoniae Beta- hemolysis – Streptococcus pyogenes Utilization of carbohydrates Oxidative - Micrococcus Fermentative – Escherichia coli

28. Growth rate Rapid growers – Vibrio cholerae Slow growers – Mycobacterium tuberculosis Pigment production Pigment producer – Staphylococcus aureus Pigment non-producer – Escherichia coli

29. Based on nutrition Autotrophs Heterotrophs Based on environmental factors Temperature Oxygen dependence pH Salt concentration Atmospheric pressure

30. Temperature Psychrophiles (15-20 C) – Pseudomonas fluorescens Mesophiles (20-40 C) – Escherichia coli , Salmonella enterica , Staphylococcus aureus Thermophiles (50-60 C)- Bacillus stearothermophilus Extremely thermophiles (as high as 250 C)

31. Oxygen dependence Aerobe (grow in ambient temperature, which contains 21% O 2 and a small amount of CO 2 , 0.03%) Obligate aerobes – Strictly require O 2 for their growth ( Pseudomonas aeruginosa ) Microaerophilic (grow under reduced O 2 , 5-10% and increased CO 2 , 8-10%)- Campylobacter jejuni , Helicobacter pylori Anaerobic bacteria use inorganic substances other than oxygen as a final electron acceptor e.g.  Pseudomonas   and Bacillus

32. pH Acidophiles ( Lactobacillus acidophilus ) Alkaliphiles ( Vibrio ) Neutralophiles (pH 6-8) Majority of the medically important bacteria grow best at neutral or slightly alkaline reaction (pH 7.2-7.6)

33. Salt concentration Halophiles e.g. Dunaliella salina Non- halophiles

34. Osmotic pressure Bacteria are about 80-90% water; they require moisture to grow Bacteria in hypertonic media causes water loss by osmosis and cell wall separates from cell membrane called as plasmolysis Bacteria in hypotonic media causes water to enter inside the cell called as osmotic lysis

35. L-Forms L-form bacteria , also known as L-phase bacteria , L-phase variants , and cell wall-deficient (CWD) bacteria , are strains of bacteria that lack cell walls . They were first isolated in 1935 by Emmy Klieneberger -Nobel , who named them "L-forms" after the Lister Institute in London Two types unstable L-forms stable L-forms

36. Bacterial reproduction Bacteria usually reproduce asexually using the process of binary fission

37. Sexual Reproduction Some bacteria reproduce sexually by exchanging some of their DNA through a conjugation tube to another bacterium Usually plasmid DNA, not genomic DNA

38. Bacterial Growth Lag Phase : bacteria adjusting to new environment and growing slowly Log Phase : exponential growth Stationary Phase : Bacteria have reached the carrying capacity of the environment Death Phase : logarithmic death of bacteria as nutrients get used up

39. Bacterial pathogenicity Capsule Capsule production is one of the major virulence factors utilised by bacteria to evade clearance from an infectious site The capsule provides bacteria with protection from the host immune response as well as antibiotics Some capsules have immunomodulatory effects The capsule protects bacteria from phagocytosis by not allowing opsonising antibodies to be recognized by phagocytic host defense cells

40. Bacterial Pathogenicity The most notorious species of bacteria that produce capsules are Streptococcus pneumoniae ( pneumococcus ), Neisseria meningitidis ( meningococcus ), and Pseudomonas aeruginosa

41. CELL WALL The cell wall of both Gram positive and Gram negative bacteria contain toxic components that are potent virulence factors and have central roles in the pathogenesis of bacterial septic shock Toxic component acts via initiation of inflammatory response by release of cytokines and interleukin-1 and activation of cascad system

42. Toxins Delivered to eukaryotic cells by S ecretion into the surrounding area D irect injection into the host cell cytoplasm via type III secretion systems Bacterial exotoxins are (1 ) A-B toxins (2) proteolytic toxins, (3) pore forming toxins ( 4) other toxins (Wilson et al ;2002)

43. Toxins A-B toxins producing bacteria are P. aeruginosa , E coli, Vibrio cholerae , Corynebacterium diphtheriae and Bordetella pertussis A subunit which possesses the enzymatic activity and the B subunit which is responsible for binding and delivery of the toxin into the host cell ( Wilson et al;2002)

44. Toxins Proteolytic toxins produced from Clostridium botulinum , Clostridium tetani and P aeruginosa Membrane-disrupting toxins are arginine (R) and threonine (T) It causes cell lysis Many Gram positive bacteria contain a sulfhydryl activated cytolysin E.g. listeriolysin O that is necessary for the escape of Listeria monocytogenes from the phagosome (Wilson et al ; 2002 )

45. Bacterial pathogenicity Adhesions Factors that bind to molecules on various host tissue cells and render the microbe resistant to these mechanical washing forces Initiate its specific biochemical reaction causes disease including proliferation, toxin secretion, host cell invasion, and activation of host cell cascades Microbial adherence factors are called adhesins (Wilson et al ; 2002)

46. Two types :-1) polypeptide 2) polysaccharide Polypeptide :- a) fimbrial b) afimbrial Gram negative bacterial pathogens has fimbriae for adherence E.g . E coli , V cholerae , P aeruginosa , and Neisseria species (Wilson et al ; 2002 )

47. Gram positive ( Staphylococcus spp , Streptococcus spp ) and mycobacterial pathogens express afimbrial adhesions Invasion P athogens gain deeper access into the host to perpetuate the infection cycle called as invasion (Wilson et al ; 2002)

48. Two types:- Extracellular and Intracellular Extracellular invasion Occurs when a microbe breaks down the barriers of a tissue to disseminate in the host while remaining outside of host cells E.g. b- haemolytic Streptococcus and S aureus (Wilson et al ; 2002)

49. Intracellular invasion Occurs when a microbe actually penetrates the cells of a host tissue and survives within this environment E.g . all gram positive and negative bacteria Target cells are both phagocytic and non phagocytic (Wilson et al ; 2002)

50. Intracellular Lifestyles Bacterial pathogens have evolved to survive and replicate within host cells after invasion Cell has killing mechanism i.e by:- Lowering the pH of bacteria Production of oxidative intermediates Activation of degradative proteases Intercellular niche for bacteria Within acidic environment (Wilson et al ; 2002)

51. 2. Inside a vacuole e.g. Coxiella burnetti 3. In host cell cytosole e.g. Shigella and Listeria utilise a pathway of cell-to-cell spread in which infection is spread from one cell to adjacent cell Bacteria residing in macrophages and neutrophils may use these cells as vehicles to spread systemically via the blood or lymphatic circulatory systems E.g. Salmonella typhi , Yersinia spp , and Brucella (Wilson et al ; 2002)

52. Virulence factors The ability of an agent of infection to produce disease is called as virulence The virulence of a microorganism is a measure of the severity of the disease it causes (Adams et al ; 2014)

53. Virulence factors

54. Virulence factors help bacteria invade the host cause disease evade host defenses Include: Attachment (via adhesins ) Colonization Invasiveness Toxins & Enzymes Inhibition of Phagocytosis (Adams et al ; 2014)

55. Attachment They allow bacteria to bind to host cells : fimbriae some bacterial cell walls capsules These adhesins bind to specific epithelium receptors or they are able to maintain even closer contact Bordetella bronchiseptica   adhesins include; fimbriae , filamentous haemagglutininadhesin (FHA) and pertactin (Adams et al ; 2014)

56. Colonization The Ability to Adhere to Host Cells and Resist Physical Removal or the establishment of the pathogen at the appropriate portal of entry Pathogens usually colonize host tissues that are in contact with the external environment E.g. Helicobacter species counter the low pH of the stomach by producing urease (Adams et al ; 2014)

57. Virulence Factors that Promote Bacterial Colonization Using Pili ( fimbriae ) to Adhere to Host Cells Using Adhesins to Adhere to Host Cells Using Biofilms to Adhere to Host Cells (Adams et al ; 2014)

58. Invasiveness T he ability of a pathogen to invade tissues Invasiveness (1) Mechanisms for colonization (adherence and initial multiplication), (2) Production of extracellular substances (" invasins "), that promote the immediate invasion of tissues (3) A bility to bypass or overcome host defense mechanisms which facilitate the actual invasive process (Adams et al ; 2014)

59. Invasiveness E.g. The intermalin surface proteins found on  Listeria monocytogenes   helps to invade mammalian cells via transmembrane proteins (Adams et al ; 2014)

60. Toxins &Enzyme They are products of a pathogen that destroy/damage/inactivate one or more vital component of the host . Classes of toxins Neurotoxins Enterotoxins Cytotoxins Toxins (Adams et al ; 2014)

61. Toxins The ability to produce toxins is known as toxogenesis Two main forms of toxin Lipopolysaccharides (primarily associated with the outer cell membrane structure of Gram-negative bacteria) and proteins( exotoxins ) Cell associated toxins (such as the lipopolysaccharides which are bound to the outer membrane) ( endotoxins )

62. Enzymes Excretion of certain pathogens to assist them in establishing infection and producing a disease There are virulence determinant enzymes that dissolve the glue between cells, thus allowing the bacteria to spread rapidly through the tissue E.g. Hyaluronidase and Coagulase

63. Virulence by spore forming bacteria Spore-forming bacteria cause some of the most significant diseases of both humans and animals e . g. Tetanus, Botulism, Gas gangrene, Anthrax, and many different enteric or gastroenteritis syndromes Virulence of this bacteria are due to production of potent protein toxins, including tetanus and botulinum toxins, anthrax toxin, and alphatoxin , epsilon-toxin (ETX), and enterotoxin (CPE) from Clostridium perfringens (Adams et al ; 2014)

64. The genes for many of these toxins and capsule production are located on plasmid E.g . Tetanus toxin plasmid, the Conjugative toxin plasmids of C. perfringens , and the pXO1 and pXO2 virulence plasmids from B. anthracis

65. Regulation of virulence factor The regulation and timing of expression of virulence factors is very important for most pathogenic bacteria Requires rapid adaptation to the new environment to allow the pathogen to colonize, survive, and grow within the host Major regulatory control mechanism by:- Sigma factor Two component system

66. Sigma factor Sigma factors are protein subunits of bacterial RNA polymerases and control the initiation of transcription at the promoter sequence Regulates prokaryotic gene expression Helps to control initiation specificity at different promoter site R egulate the expression of genes in response to stationary phase, nutrient deprivation , and oxidative and osmotic stress

67. RpoS (s38) sigma factor important for virulence in a number of bacterial pathogens,including Salmonella typhimurium , E coli, P aeruginosa RpoE (s24 ) a sigma factor which responds to periplasmic stress RpoH ( s32) a heat shock sigma factor which is important in the regulation of virulence in Vibrio cholerae

68. Two component systems A Sensor protein that is embedded in the bacterial membrane which “senses” different physiological conditions of the bacterial cell (2) A Response regulator which usually binds to the promoter region of a gene to activate or repress transcription Helps in regulation of iron, phosphate, nitrogen, carbon, capsule production, and flagellar activity

69. EVOLUTION OF BACTERIAL PATHOGENS Genetic makeup of bacterial genome is rapidly changing by “horizontal gene transfer” Horizontal gene transfer refers to the incorporation of genetic elements transferred from a donor organism directly into the genome of the recipient organism and forms pathogenicity islands E.g. E.coli , Salmonella, Vibrio spp . , Shigella spp . , Yersinia spp. , Listeria spp . , S aureus

70. Addition to pathogenicity islands, plasmids and bacteriophages can also be transferred horizontally

71. Antibiotic resistance What are antibiotics? Powerful medicines that treat bacterial infections They work by either killing bacteria or preventing growth and reproduction of bacteria Widespread use of antibiotics led to the emergence of antibiotic resistant

72. Both Gram negative and Gram positive bacteria have acquired resistance to antimicrobial drugs E.g. Shigella, Salmonella, E coli, and Enterococcus faecium Methicillin resistant S aureus , causes nosocomial infections Vanomycine resistance Enterococcus

73. How the resistance is produced? Three common type of antimicrobial resistance :- Modification of target site Altering uptake of antibiotics Inactivation of antibiotic Its occurs by two genetic process Spontaneous mutation By acquication

74. Horizontal gene transfer Genetic elements are transferred from one organism to another, intraspecies and interspecies It is transferred as mobile element i.e. Transposons or by naked uptake of DNA through transformation or by sexual transformation by conjugation or by incorporation of DNA into a phage genome

75. Antibiotic resistance E.g. multidrug resistance of S typhimurium DT104, a food borne pathogen is due to the integration of a transposon A mutation which alters the binding site of a drug would decrease antibiotic sensitivity and thus increase drug resistance E.g. multi drug resistant M tuberculosis

76. Interaction of Pathogens with the Innate Immune System Micro-organisms are exposed non-specific barriers to infection after introduction of the microbe into the host Include Epithelial cells of the skin Antimicrobial substances in secretions Complement proteins in the blood Leucocytes in the blood and tissues

77. Immune response The host defense mechanisms are mediated by the immune system   Immunity  refers to the relative state of resistance of the host to infectious disease The  immune system  is composed of two major subdivisions Innate or nonspecific immune system Adaptive or specific immune system

78. Innate immune system is a primary defense mechanism while the adaptive immune system acts as a second line of defense Both aspects of the immune system have cellular and humoral components which carry out their protective functions Cells or components of the innate immune system influence the adaptive immune system and vice versa

79. Immunity Adaptive immune system requires some time to react to an invading organism, whereas the innate immune system acts rapidly The adaptive immune system is antigen specific. The innate system is not antigen specific Adaptive immune system exhibits an immunological memory but innate immunity does not

80. Cellular defense A variety of tissue cells are involved in innate and adaptive immunity, hence the term cellular defense It has Neutrophils and Macrophages for phagocytosis Basophils and Mast cells for inflammation B cells and T cells which account for antibody mediated immunity and cell mediated immunity

81. Innate immune response They play major role by complement activation by alternative pathway specially for gram – ve bacteria By phagocytosis Release of cytokinins Activation of NK cells

82. Adaptive Immunity By production of antibodies Neutralization of bacterial toxins Mucosa protection ( IgA ) and activation of the complement by the classical pathway Activating phagocytosis by the stimulation of the Fc receptor

83. Specific Immune Responses:- depends on type of pathogens Extracellular bacteria and toxins Intracellular bacteria Encapsulated bacteria

84. References Textbook of Microbiology by Ananthanarayan and Paniker Wilson, J.W., Schurr , M.J., LeBlanc, C.L., Ramamurthy, R., Buchanan, K.L. and Nickerson, C.A., 2002. Mechanisms of bacterial pathogenicity. Postgraduate medical journal ,  78 (918), pp.216-224. Rahme , L.G., Stevens, E.J., Wolfort , S.F., Shao , J., Tompkins, R.G. and Ausubel , F.M., 1995. Common virulence factors for bacterial pathogenicity in plants and animals.  Science ,  268 (5219), pp.1899-1902. Adams, V., Li, J., Wisniewski, J.A., Uzal , F.A., Moore, R.J., McClane , B.A. and Rood, J.I., 2014. Virulence plasmids of spore-forming bacteria. Microbiology spectrum ,  2 (6). Textbook of Microbiologt by Sharma S. N. and Adalka Janeway Jr , C.A. and Medzhitov , R., 2002. Innate immune recognition. Annual review of immunology ,  20 (1), pp.197-216.