1. The Growth, Survival, & Death
of Microorganisms

2. KEY TERMS Biocide Bacteriostatic Bactericidal Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Antibiotic
Exponential growth
Growth Curve
Binary fission

3. Survival of Microorganisms in the Natural Environment
Population of microorganisms in the biosphere is roughly constant
Growth is counterbalanced by death
The survival of microbial within its niche is determined by successful competition for nutrients

4. Survival of Microorganisms in the Natural Environment
Most of our understanding of microbial physiology has come from the study of isolated cell lines growing under optimal conditions
Many microorganisms compete in the natural environment while under nutritional stress, a physiologic state quite unlike that observed in the laboratory
A vacant microbial niche in the environment will soon be filled
Public health procedures that eliminate pathogenic microorganisms by clearing their niche are likely to be less successful than methods that leave the niche occupied by successful nonpathogenic competitors.

5. The Meaning of Growth
Growth is the orderly increase in the sum of all the components of an organism
Increase in size that results when a cell takes up water or deposits lipid or polysaccharide is not true growth
Cell multiplication is a consequence of growth
In unicellular organisms, growth leads to an increase in the number of individuals

6. The Measurement of Microbial Concentrations
Cell Concentration
Turbidity of a culture, measured by photoelectric means
The viable cell count is usually considered the measure of cell concentration
Biomass Density
determining the dry weight of a microbial culture after it has been washed with distilled water
In practice, the investigator customarily prepares a standard curve that correlates dry weight with turbidity

7. Measuring bacterial mass (live + dead) in liquid culture
Turbidity
(Cloudiness)

8. Measuring viable bacteria
Colony forming units
colony

9. Bacteria Multiplication
Binary fission
G-菌分裂

10. The Growth Curve A: The Lag Phase E: The Maximum Stationary Phase
C: The Exponential Phase F: The Death Phase (Decline Phase)

11. Growth Curve
Stationary
COLONY
FORMING
UNITS
Death
Log
Lag
TIME

12. Growth Curve
Stationary
TURBIDITY
(cloudiness)
Autolysis
Log
Lag
TIME

13. Generation Time Time for bacterial mass to double Example
100 bacteria present at time 0
If generation time is 2 hr
After 8 hr mass = 100 x 24

14. The Meaning of Death
For a microbial cell, death means the irreversible loss of the ability to reproduce (grow and divide)
The empirical test of death is the culture of cells on solid media: A cell is considered dead if it fails to give rise to a colony on any medium
Obviously, the reliability of the test depends upon choice of medium and conditions
Methods
Sterilization
Antimicrobials

15. Antimicrobial Agents Biocide Bacteriostatic Bactericidal Sterilization
The following terms are commonly employed in connection with antimicrobial agents and their uses
Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics

16. Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics
A general term describing a chemical agent, usually broad-spectrum, that inactivates microorganisms

17. Bacteriostatic Biocide Bactericidal Sterilization Disinfectant Septic
Antiseptic
Aseptic
Reservation
Antibiotics
A specific term referring to the property by which a biocide
is able to inhibit bacterial multiplication; multiplication
resumes upon removal of the agent
The terms “fungistatic” and “sporostatic” refer to biocides that inhibit the growth of fungi and spores, respectively

18. Bactericidal Biocide Bacteriostatic Sterilization Disinfectant Septic
Antiseptic
Aseptic
Reservation
Antibiotics
A specific term referring to the property by which a biocide is able to kill bacteria
In some cases, the agent causes lysis of the cells; in other cases, the cells remain intact and may continue to be metabolically active
Fungicidal
Sporicidal
Virucidal

19. Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics
A physical or chemical process that completely destroys or removes all microbial life, including spores.

20. There are 4 major methods for sterilization:
1. Heat: Autoclaving which involves heating liquids (e.g. media) or solids to 121oC under steam pressure. However, the materials must be heat resistant.
2. Ethylene oxide is sometimes used in hospitals for equipment that cannot be heated.
3. Membrane filters have pores that trap bacteria, but allow drugs and small chemicals to pass through; thus pre-sterilized filters can be used to sterilize delicate solutions.
4. Radiation
UV light is used for decreasing bacterial levels on surfaces such as in operating rooms; however it is not totally effective
Ionizing radiation is more efficient and can be used for sterilizing instruments and food

21. Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics
Products or biocides used to kill microorganisms on inanimate objects or surfaces
Disinfectants can be sporostatic but are not necessarily sporicidal

22. Disinfectants
Can be used in killing many bacteria on certain instruments, but cannot be used for internal consumption or on skin
(e.g. phenol-based products)

26. Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics
Characterized by the presence of pathogenic microbes in living tissue

27. Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics
A biocide or product that destroys or inhibits the growth of microorganisms in or on living tissue

28. Antiseptics
These products are used topically (e.g. on skin surfaces) to reduce bacterial load.
(e.g. iodine or 70% alcohol)

29. Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics
Characterized by the absence of pathogenic microbes

30. Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics
The prevention of multiplica-tion of microorganisms in formulated products, including pharmaceuticals and foods

31. Biocide
Bacteriostatic
Bactericidal
Sterilization
Disinfectant
Septic
Antiseptic
Aseptic
Reservation
Antibiotics
Naturally occurring or synthetic organic compounds which inhibit or destroy selective bacteria, generally at low concentrations

32. ANTIBIOTICS Antibiotics are agents that are "selectively"
toxic for bacteria (either killing them [bactericidal] or inhibiting their growth [bacteriostatic]) without harm to the patient.

33. Principles and Definitions
Selectivity
Selectivty Toxicity
Antibiotics must act on structures found in bacteria, but not in the host
Therapeutic index
Toxic dose/ Effective dose
Categories of antibiotics
Bactericidal
Usually antibiotic of choice
Bacteriostatic
Duration of treatment sufficient for host defenses

34. Principles and Definitions
Antibiotic susceptibility testing (in vitro)
Minimum inhibitory concentration (MIC)
Lowest concentration that results in inhibition of visible growth
Minimum bactericidal concentration (MBC)
Lowest concentration that kills 99.9% of the original inoculum

35. Antibiotic Susceptibility Testing
Chl
Amp
Ery
Str
Tet
Disk Diffusion Test 8 4 2 1
Tetracycline (ug/ml)
MIC = 2 ug/ml
Determination of MIC

36. Principles and Definitions
Combination therapy
Prevent emergence of resistant strains
Temporary treatment until diagnosis is made
Antibiotic synergism
Penicillins and aminoglycosides
CAUTION: Antibiotic antagonism
Penicillins and bacteriostatic antibiotics
Antibiotics vs chemotherapeutic agents vs antimicrobials

37. Antibiotics: Mode of Action
Damage to DNA
Protein denaturation
Disruption of cell
membrane or wall
Removal of free
sulfhydryl group
Chemical antagonism

39. Antimicrobial Drug Resistance Principles and Definitions
Resistance can arise by mutation or acquisition of a plasmid with resistance gene
Resistance provides a selective advantage
Resistance can result from single or multiple steps
Cross resistance vs multiple resistance
Cross resistance -- Single mechanism-- closely related antibiotics
Multiple resistance -- Multiple mechanisms -- unrelated antibiotics

40. Antimicrobial Drug Resistance Mechanisms
Altered permeability
Altered influx
Gram negative bacteria
Altered efflux
tetracycline
Inactivation
beta-lactamase
Chloramphenicol acetyl transferase

41. Antimicrobial Drug Resistance Mechanisms
Altered target site
Penicillin binding proteins (penicillins)
RNA polymerase (rifampin)
30S ribosome (streptomycin)
Replacement of a sensitive pathway
Acquisition of a resistant enzyme (sulfonamides, trimethoprim)

42. Antibiotics: Inhibitors of Cell Wall Synthesis

43. INHIBITORS OF CELL WALL SYNTHESIS
One major class of antibiotics inhibit the synthesis of peptidoglycan
Once cell wall synthesis (involving penicillin binding proteins) is inhibited, enzymatic autolysis of the cell wall can occur

44. Without the restraining influence of the cell
wall the high osmotic pressure inside the cell
bursts the inner and/or outer membranes of
bacteria.
Antibiotics which are inhibitors of cell wall
biosynthesis are generally bactericidal

45. Mechanisms involved in the inhibition of peptidoglycan synthesis
Cycloserine (an anti-mycobacteria drug)
The terminal two amino acids of a peptide side chain of
peptidoglycan are unusual amino acids (D-alanine as
opposed to its isomer L-alanine)
The antibiotic cycloserine is an analog of D-alanine and
interferes with enzymatic conversion of L-alanine to
D-alanine in the cytoplasm. Thus subsequent synthesis of
peptidoglycan cannot occur

46. 2. Bacitracin
The peptidoglycan subunit (containing one side-chain and
an attached peptide to be used in cross-bridge formation) is
passed across the cytoplasmic membrane attached to
undecaprenol diphosphate.
After the nascent peptidoglycan monomer leaves the carrier
on reaching the cell wall, the undecaprenol diphosphate is
dephosphorylated to its monophosphate form.
Bacitracin inhibits the dephosphorylation reaction and in
the absence of monophosphorylated carrier peptidoglycan
subunit synthesis stops.

47. 3. Vancomycin
The final step in peptidoglycan synthesis involves linking
the sugar portion of the peptidoglycan subunit to the glycan
backbone of the existing cell wall polymer.
Cross-linking of the peptide portion of the subunit to a
peptide in the cell wall then occurs.
During this process D-alanine is enzymatically excised from
the end of a pre-existing peptide side chain allowing it to
be cross-linked (by a new peptide bond) to the recently
synthesized peptidoglycan subunit.
Vancomycin binds to D-alanine-D-alanine thus sterically
inhibits transpeptidation (cross-linking).

48. 4. beta lactam The beta lactam antibiotics include penicillins
(e.g. ampicillin), cephalosporins and monobactams.
They bind to and inhibit enzymes (penicillin binding
proteins) involved in the transpeptidation (cross-linking) of
peptidoglycan. These antibiotics have in common the four
membered lactam ring.
Attached to the lactam, penicillins have an additional five
membered ring and cephalosporins a six membered ring

49. STRUCTURE OF PENICILLINNH CH O N
CH3
COOH S
Site of penicillinase action,
Breakage of the beta-lactam ring

50. Many penicillins display little activity against Gram
negative bacteria, since they do not penetrate the
outer membrane
Cephalosporins and other newer penicillins are active against Gram negative bacteria, since they can penetrate the outer membrane

51. Penicillins can be destroyed by beta lactamase
(penicillinase) produced by resistant bacterial strains
Clavulinic acid, also has a beta lactam component which
binds strongly to beta lactamases inhibiting their activity
Another form of resistance involves a change in the
structure of penicillin binding proteins such that the
antibiotic does not bind efficiently

54. POLYMYXIN B
Polymyxin B binds to the lipid A portion of lipopolysaccharide
and also to phospholipids
This disrupts the outer membrane of Gram negative bacteria
Since the cell membrane is not exposed in Gram positive
bacteria, polymyxin has little activity against them
This drug is toxic to human cells, since it can also lyse
eukaryotic membranes; this explains its limited clinical use

55. STRUCTURE OF POLYMYXIN

56. Antibiotics: Protein Synthesis, Nucleic Acid Synthesis and Metabolism

57. Review of Initiation of Protein Synthesis1 3 2
GTP
Initiation Factors
mRNA 3 1 2
GTP
30S Initiation Complex
f-met-tRNA
Spectinomycin 1 2
GDP + Pi
70S
70S Initiation Complex A P
Aminoglycosides

58. Review of Elongation of Protein Synthesis
GTP A P Tu
GDP Ts + Pi
Tetracycline A P
Chloramphenicol G
GTP
GDP + Pi A P
Fusidic Acid
Erythromycin

59. Protein Synthesis Inhibitors
Mostly bacteriostatic
Selectivity due to differences in prokaryotic and eukaryotic ribosomes
Some toxicity - eukaryotic 70S ribosomes

60. Antimicrobials that Bind to the 30S Ribosomal Subunit

61. Aminoglycosides (bactericidal) streptomycin, kanamycin, gentamicin, tobramycin, amikacin, netilmicin, neomycin (topical)
Mode of action - The aminoglycosides irreversibly bind to the 16S ribosomal RNA and freeze the 30S initiation complex (30S-mRNA-tRNA) so that no further initiation can occur. They also slow down protein synthesis that has already initiated and induce misreading of the mRNA. By binding to the 16 S r-RNA the aminoglycosides increase the affinity of the A site for t-RNA regardless of the anticodon specificity. May also destabilize bacterial membranes.
Spectrum of Activity -Many gram-negative and some gram-positive bacteria; Not useful for anaerobic (oxygen required for uptake of antibiotic) or intracellular bacteria.
Resistance - Common
Synergy - The aminoglycosides synergize with $-lactam antibiotics. The beta-lactams inhibit cell wall synthesis and thereby increase the permeability of the aminoglycosides.

62. Tetracyclines (bacteriostatic) tetracycline, minocycline and doxycycline
Mode of action - The tetracyclines reversibly bind to the 30S ribosome and inhibit binding of aminoacyl-t-RNA to the acceptor site on the 70S ribosome.
Spectrum of activity - Broad spectrum; Useful against intracellular bacteria
Resistance - Common
Adverse effects - Destruction of normal intestinal flora resulting in increased secondary infections; staining and impairment of the structure of bone and teeth.

63. Spectinomycin (bacteriostatic)
Mode of action - Spectinomycin reversibly interferes with m-RNA interaction with the 30S ribosome. It is structurally similar to the aminoglycosides but does not cause misreading of mRNA.
Spectrum of activity - Used in the treatment of penicillin-resistant Neisseria gonorrhoeae
Resistance - Rare in Neisseria gonorrhoeae

64. Antimicrobials that Bind to the 50S Ribosomal Subunit

65. Chloramphenicol, Lincomycin, Clindamycin (bacteriostatic)
Mode of action - These antimicrobials bind to the 50S ribosome and inhibit peptidyl transferase activity.
Spectrum of activity - Chloramphenicol - Broad range; Lincomycin and clindamycin - Restricted range
Resistance - Common
Adverse effects - Chloramphenicol is toxic (bone marrow suppression) but is used in the treatment of bacterial meningitis.

66. Macrolides (bacteriostatic) erythromycin, clarithromycin, azithromycin, spiramycin
Mode of action - The macrolides inhibit translocation.
Spectrum of activity - Gram-positive bacteria, Mycoplasma, Legionella
Resistance - Common

67. Antimicrobials that Interfere with Elongation Factors
Selectivity due to differences in prokaryotic and eukaryotic
elongation factors

68. Fusidic acid (bacteriostatic)
Mode of action - Fusidic acid binds to elongation factor G (EF-G) and inhibits release of EF-G from the EF-G/GDP complex.
Spectrum of activity - Gram-positive cocci

69. Inhibitors of Nucleic Acid Synthesis

70. Inhibitors of RNA Synthesis
Selectivity due to differences between prokaryotic and eukaryotic
RNA polymerase

71. Rifampin, Rifamycin, Rifampicin, Rifabutin (bactericidal)
Mode of action - These antimicrobials bind to DNA-dependent RNA polymerase and inhibit initiation of mRNA synthesis.
Spectrum of activity - Wide spectrum but is used most commonly in the treatment of tuberculosis
Resistance - Common
Combination therapy - Since resistance is common, rifampin is usually used in combination therapy.

72. Inhibitors of DNA Synthesis
Selectivity due to differences between prokaryotic and eukaryotic enzymes

73. Quinolones (bactericidal) nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, lomefloxacin, sparfloxacin
Mode of action - These antimicrobials bind to the A subunit of DNA gyrase (topoisomerase) and prevent supercoiling of DNA, thereby inhibiting DNA synthesis.
Spectrum of activity - Gram-positive cocci and urinary tract infections
Resistance - Common for nalidixic acid; developing for ciprofloxacin

74. Antimetabolite Antimicrobials

75. Inhibitors of Folic Acid Synthesis
Basis of Selectivity
Folic Acid Metabolism
p-aminobenzoic acid + Pteridine
Dihydropteroic acid
Dihydrofolic acid
Tetrahydrofolic acid
Pteridine synthetase
Dihydrofolate synthetase
Dihydrofolate reductase
Thymidine
Purines
Methionine
Sulfonamides
Trimethoprim

76. Sulfonamides, Sulfones (bacteriostatic)
Mode of action - These antimicrobials are analogues of para-aminobenzoic acid and competitively inhibit formation of dihydropteroic acid.
Spectrum of activity - Broad range activity against gram-positive and gram-negative bacteria; used primarily in urinary tract and Nocardia infections.
Resistance - Common
Combination therapy - The sulfonamides are used in combination with trimethoprim; this combination blocks two distinct steps in folic acid metabolism and prevents the emergence of resistant strains.

77. Trimethoprim, Methotrexate, Pyrimethamine (bacteriostatic)
Mode of action - These antimicrobials binds to dihydrofolate reductase and inhibit formation of tetrahydrofolic acid.
Spectrum of activity - Broad range activity against gram-positive and gram-negative bacteria; used primarily in urinary tract and Nocardia infections.
Resistance - Common
Combination therapy - These antimicrobials are used in combination with the sulfonamides; this combination blocks two distinct steps in folic acid metabolism and prevents the emergence of resistant strains.

78. Anti-Mycobacterial Antibiotics

79. Para-aminosalicylic acid (PSA) (bacteriostatic)
Mode of action - Similar to sulfonamides
Spectrum of activity - Specific for Mycobacterium tuberculosis

80. Dapsone (bacteriostatic)
Mode of action - Similar to sulfonamides
Spectrum of activity - Used in treatment of leprosy (Mycobacterium leprae)

81. Isoniazid (INH) (bacteriostatic )
Mode of action - Isoniazid inhibits synthesis of mycolic acids.
Spectrum of activity - Used in treatment of tuberculosis
Resistance - Has developed

82. Cultivation of Microorganisms
Section I Fundaments of Microbiology
Cultivation of Microorganisms
Zhao-Hua Zhong, Ph.D., Prof.
Department of Microbiology
Harbin Medical University

83. KEY TERMS Obligate aerobe Obligate anaerobe Aerotolerant anaerobe
Facultative anaerobe
Microaerophilic
Siderophore
Mesophile
Thermophile
Psychrophile
Generation time
Growth curve
Glycolysis
Fermentation
Anaerobic respiration
Aerobic respiration
Tricarboxylic acid (TCA)
cycle or Krebs cycle
Oxidative phosphorylation
Ubiquinone
Glyoxylate pathway

84. Bacterial Requirements for Growth
Energy
Oxygen (or absence)
Nutrients
Optimal temperature
Optimal pH

85. SOURCES OF METABOLIC ENERGY
The three major mechanisms for generating metabolic energy are
Fermentation
Respiration
Photosynthesis
At least one of these mechanisms must be employed if an organism is to grow

86. Fermentation
The formation of ATP in fermentation is not coupled to the transfer of electrons
Fermentation is a substrate phosphorylation, an enzymatic process in which a pyrophosphate bond is donated directly to ADP (adenosine diphosphate) by a phosphorylated metabolic intermediate
The phosphorylated intermediates are formed by metabolic rearrangement of a fermentable substrate such as glucose, lactose, or arginine
Example: fermentation of a molecule of glucose (C6H12O6) yields a net gain of two pyrophosphate bonds in ATP and produces two molecules of lactic acid (C3H6O3)

87. Respiration
Respiration is chemical reduction of an oxidant (electron acceptor) through a specific series of electron carriers in the membrane establishes the proton motive force across the bacterial membrane
Reductants (electron donor)
may be organic or inorganic: For example, lactic acid serves as a reductant for some organisms, and hydrogen gas is a reductant for other organisms
Oxidants
Gaseous oxygen (O2) often is employed as an oxidant, but alternative oxidants that are employed by some organisms include carbon dioxide (CO2), sulfate (SO42–), and nitrate (NO3−)

88. Photosynthesis
Photosynthesis is similar to respiration in that the reduction of an oxidant via a specific series of electron carriers establishes the proton motive force
Difference in the two processes
in photosynthesis the reductant and oxidant are created photochemically by light energy absorbed by pigments in the membrane
Photosynthesis can continue only as long as there is a source of light energy

89. Obligate Aerobes No fermentation Oxidative phosphorylation
Grow in presence of oxygen
No fermentation
Oxidative phosphorylation

90. Obligate Anaerobes No oxidative phosphorylation Fermentation
Killed by oxygen
Lack certain enzymes
superoxide dismutase
O2-+2H H2O2
catalase
H2O H20 + O2
peroxidase
H2O H20 /NAD NADH

91. Facultative Anaerobes
Fermentation
Aerobic respiration
Survive in oxygen

92. Microaerophilic Bacteria
Grow
low oxygen
Killed
high oxygen

93. Optimal Growth Temperature
Mesophile
human body temperature
pathogens
opportunists
Pyschrophile
close to freezing
Thermophile
close to boiling

94. pH
Many grow best at neutral pH
Some can survive/grow
- Acid
Alkali

95. Nutrient Requirements
Carbon
Nitrogen
Phosphorus
Sulfur
Metal ions (e.g. iron)