Nutrition, Growth, and Metabolism

1. Nutrition, Growth, and Metabolism

2. In Medical Microbiology, microbial cultivation is required for the following purposes • In Medicine • 1-Diagnosis of most infectious diseases • 2-Selection of drug of choice ( antibiotics ) for treatment of infection • In other fields • 3-Preparation of Vaccine • 4-Research tool in molecular genetics

3. Microbial growth requirements • What are the chemical growth factors required for isolation of microbes in vitro? • 1-Carbon • Organic source Glucose • Inorganic source CO2 • 2-Nitrogen • Organic source Protein • Inorganic source Atmospheric nitrogen • Autotrophic microbes using inorganic carbon and nitrogen • Heterotrophic microbes using organic carbon and nitrogen • Other chemical requirements • 3- Hydrogen, Oxygen, Phosphorus, Carbon, Nitrogen, Ions, and Sulfur

4. Physical growth factors • 1- pH and Buffer requirements • Pathogenic bacteria grow best at neutral pH which is typically between pH (6.8 to 7.4) • Acidophilic bacterium is able to survive with acidic pH • Example: Helicobacter pylori • On the other extreme, bacteria that prefer alkaline conditions are known as alkaliphiles • Example: Vibrio cholerae • Fungi such as yeasts and molds; grow best at acidic conditions ( pH 5 )

5. 2- Salts concentration • A specific concentration of NaCl is required for microbial growth in vitro • It is equal to normal saline salts concentration (0.9% NaCl) • Halophilic bacteria resist high salt concentration

6. 3- Temperature requirements • Mesophiles • grow at optimum of 37 ᵒC • human body temperature • Pathogens • Opportunists • Pyschrophile • close to freezing • Thermophile • close to boiling

7. 4- Gaseous requirements and Humidity • According to oxygen requirements, microbes can be divided into the following groups • 1- Strict or Obligate Aerobe • O2 must be present to grow • Example: Mycobacterium sp. • 2- Strict or Obligate Anaerobes • Oxygen must be absent • Killed by oxygen • Example: Clostridium sp.

8. 3- Facultative anaerobes • Adaptable organisms that use oxygen when present but can switch to anaerobic pathways in its absence • Survive in the presence of O2 • Example: Escherichia coli • 4- Microaerophilic • Only use low concentrations of oxygen ( around 5%) • Example : Helicobacter pylori • Grow in low oxygen • Killed in high oxygen

9. The Bacterial Growth Curve • Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission • Bacterial growth curve represents the relationship between microbial quantity and time of incubation • During Lag phase, bacteria adapt themselves to growth conditions (number of dividing cell is zero) • Exponential phase ( Log phase): is a period characterized by Rapid cell doubling • Doubling time can be as short as twenty minutes or as long as several days

10. During stationary phase, the growth rate slows as a result of nutrient depletion and accumulation of toxic products • This phase is a constant value as the rate of bacterial growth is equal to the rate of bacterial death • At death phase, bacteria run out of nutrients and die

11. Bacterial binary fission

12. Bacterial growth curve

13. Microbial Metabolism • According to biochemical pathway used in energy production, bacterial metabolism can be categorized into three types: • 1-Aerobic Respiration • Molecular oxygen serves as the final electron acceptor • 38 ATP molecules will be produced by oxidation of one glucose molecule • Used by obligatory aerobic bacteria for energy production • such as: Mycobacterium sp.

14. 2-Anaerobic Respiration • Inorganic sulfate or nitrate act as the final electron acceptor • 38 ATP molecules will be produced by catabolism of one glucose molecule • Used by obligatory anaerobic bacteria such as: Clostridium sp. • 3-Fermentation • Lactic acid ( produced by bacteria) or ethanol ( produced by yeast) serves as final electron acceptor • Only 2 ATP molecules will be produced by fermentation of one glucose molecule • Used by facultative anaerobic bacteria such as : E. coli

15. Microbial Genetics • Prokaryotic Genome • Most prokaryotic genes are carried on the bacterial chromosome, a single circle of DNA • Many bacteria contain additional genes on plasmids • Plasmid is an extra-circular supercoiled DNA that carry some important genes such as the antibiotics resistance genes • Both bacteria chromosome and plasmid are called replicons

16. Genetics is the study of inheritance and variation • Genetic information encoded in DNA • Function of genetic material • 1- Replication of the genome • 2- Expression of DNA to mRNA then to protein

17. Definitions • Genotype • the complete set of genetic determinants of an organism • Phenotype • expression of specific genetic material

18. Bacterial DNA • 2 types of DNA in bacteria • Chromosomal • Extra-chromosomal (plasmid)

19. Plasmid • Extrachromosomal DNA • Found in most species of bacteria. • Govern their own replication • Genetic exchange, amplify genes • Transfer by conjugation • Code for resistance to antibiotics & toxins

20. Bacterial Structure

21. Bacterial Structure

22. Gene Transfer • Transfer of DNA among prokaryotes is widespread between different strains of same bacterial species • Mechanisms of Gene Transfer • 1- Conjugation • 2- Transduction (is a phage-mediated genetic transfer) • 3- Transformation

23. Bacterial conjugation • Conjugation: is a mechanism of gene transfer by which plasmids will be transferred from one bacterial cell to another by a mean of Sex pili • Hospital-dwelling bacteria resist antibiotics due to conjugation

24. Conjugation

25. Transduction • Transduction is the process by which DNA is transferred from one bacterium to another by a virus • It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector

26. Transduction

27. Bacterial transformation • A stable genetic change brought about by the uptake of naked DNA and competence refers to the state of being able to take up exogenous DNA from the environment • There are two forms of transformation and competence: natural and artificial

28. The Microbial Virulence factors • Virulence factors are external cellular structures, enzymes, and toxins that enhance microbial pathogenicity • In general, the most important virulence factors are: • 1-Microbial capsule • Microbe resist host acidic environment (stomach gastric acid) • Microbe resist host proteolyticenzyme • (Present in Saliva, and stomach) • Microbe resist phagocytosis

29. 2-Fimbriae or Pili • Microbial adhesion to the host cell surface • Adhesion could be also enhanced by receptor-antigen interaction • 3-Microbial Enzymes • Collagenase enzyme • enhances microbial invasion; due to degradation of extracellular matrix components • Urease • Neutralization of acidic pH ( urine, stomach) • Coagulase • Catalase have different functions

30. 4-Bacterial Toxins • A-Exotoxins • Well known poisonous substances. • Chemical nature • Proteins (two polypeptide components) • Almost all are Heat-labile at 60 ˚C • Intracellular toxin fraction could • 1-Inhibit cellular protein biosynthesis • 2-Cause ionic imbalance and loss of water • 3-Inhibit the release of neurotransmitters

31. B-Endotoxins • Chemical nature • Lipopolysaccharide, the component of Gram’s negative bacterial outer membrane • Heat-Stable at 100 ˚C • 5-The microbial Hemolysin • Degradation of RBCs, Hemoglobin and NADH will be released • 6-The microbial Haemagglutinine and Coagulase enzyme • Agglutination of RBCs; the microbe escapes Humoral immunity

32. 7-The microbial Beta-Lactamases • Some microbes have ability to resist antibiotics due to production of Beta-Lactamase enzymes • Some strains of Staphylococcican hydrolyze the Beta-Lactam ring shown

34. In the absence of β-lactam antibiotics, the bacterial cell wall plays an important role in bacterial reproduction.

35. Adding β-lactam antibiotics to the cell medium while bacteria are dividing will cause them to shed their cell walls and fail to divide, forming large, fragile spheroplasts.