Download Microbial Metabolism

Microbial Metabolism
Biochemical tests
Zivuku.M
Content
• Microbial metabolism
(anabolism and catabolism)
• Microbial biochemical tests
(carbohydrates fermentation tests, Methyl red
test, VP test, Indole test, Citrate test)
• Microbial products
(pyrogen's, antibiotics, vitamins, bacteriocins)
Microbial Metabolism
Metabolism refers to all the biochemical reactions that occur in a
cell or organism.
1) pathways for the interconversion of focal metabolites
2) assimilatory pathways for the formation of focal metabolites
3) biosynthetic sequences for the conversion of focal metabolites to
end products
4) pathways that yield metabolic energy for growth and
maintenance.
Microbial Metabolism
The key concepts of glycolysis, Krebs Cycle, oxidative
phosphorylation have been or will be discussed in biochemistry.
Concentrates here are put on the products of bacterial metabolism
with medical importance (e.g.
bacterial pathogenesis).
for laboratory diagnosis or for
•
Medical Important Metabolic Products
1. anabolic processes
(synthesis or build up)
2. catabolic processes
(decomposition or break down)
Catabolic Products and Biochemical Testing
a. Carbohydrate Fermentation Tests
Medium: Carbohydrate fermentation broth with a
Durham tube (a small inverted vial filled with the
carbohydrate fermentation broth).
If gas is produced during fermentation of the sugar, it is
trapped at the top of the Durham tube and appears as a
bubble.
pH indicator: phenol red
a. Carbohydrate Fermentation Tests
Principle: Because the type of enzyme(s) produced by
bacteria is genetically controlled, the fermented pattern of
sugars may be unique to a species, but may be different
between different species.
Fermentation products are usually acid (lactic acid, acetic
acid etc.), neutral (ethanol etc.) or gases (carbon dioxide,
hyrogen, etc).
a. Carbohydrate Fermentation Tests
Result:
positive
yellow color
yellow color with gas bubble
negative
red color, no gas bubble
b. Methyl Red (MR) Test
Medium: contains glucose and peptone
Indicator: methyl Red
All enterics oxidize glucose for energy; however the end
products vary depending on bacterial enzymes.
b. Methyl Red (MR) Test
For bacteria (e.g. E. coli ) that produces acids, causing the pH to
drop below 4.4. When the pH indicator methyl red is added to this
acidic broth it will be cherry red (a positive MR test).
For bacteria (e.g. Klebsiella and Enterobacter) that produce more
neutral products from glucose (e.g. ethyl alcohol, acetyl methyl
carbinol). In this neutral pH the growth of the bacteria is not
inhibited. The bacteria thus begin to attack the peptone in the broth,
causing the pH to rise above 6.2. At this pH, methyl red indicator is
a yellow color (a negative MR test).
Result:
c. Voges-Proskauer (VP) Test
Medium: contains glucose and peptone
Indicator: Barritt's A (alpha-napthol) and Barritt's B (potassium
hydroxide)
When these reagents are added to a broth in which acetyl methyl
carbinol is present, they turn a pink-burgundy color (a positive
VP test).
This color may take 20 to 30 minutes to develop.
E. coli does not produce acetyl methyl carbinol, but Enterobacter
and Klebsiella do.
c. Voges-Proskauer (VP) Test
d. Citrate Utilization Test
The citrate test utilizes Simmon's citrate
media to determine if a bacterium can grow
utilizing citrate as its sole carbon and
energy source.
Growth of bacteria in the media leads to
development of a Prussian blue color
(positive citrate).
e. Indole Test
The test organism is inoculated into tryptone broth, a rich
source of the amino acid tryptophan.
Indole positive bacteria such as E. coli produce tryptophanase,
an enzyme that cleaves tryptophan, producing indole and other
products.
When Kovac's reagent is added to a
broth with indole in it, a dark pink
color develops.
Indol test
Methyl test
VP test
Citrate utilization test
I M Vi C test
E. coli
C. perfringens
I
M
Vi C
+
—
+
—
—
+
—
+
• f. Hydrogen Sulfide (H2S ) formation
To determine the ability of a bacterium to produce hydrogen
sulfide (H2S) by enzymatic reaction on amino acids such as
cysteine, cystine and methionine.
The hydrogen sulfide combines with ferrous
sulfide (Fe2S) in the triple sugar iron (TSI)
agar to form a black to dark insoluble
precipitate.
• g. Urease Test
Medium: urea agar slant
Indicator: phenol red
Principle: The hydrolysis of urea by urease produces
ammonia and carbon dioxide. The formation of ammonia
alkalinizes the medium, and the pH is detected by the color
change from light orange to pink-red.
Positive result: pink-red color
Negative result: light orange
What are the medically important
anabolic products of bacteria?
Synthetic Products
• Pyrogens
• Toxins and Invasive Enzymes
• Antibiotics
• Vitamins
• Bacteriocins
• Pigments
• Pyrogens
• the products of many bacteria, especially gram-negative
bacteria, which resulting in fever when injected into animals or
humans.
• They are polysaccharides in cell wall (G+ bacteria) or LPS
(G- bacteria).
• They are highly resistant to high heat (not being destroyed
heating at 121C for 15-20 min), but can be destroyed by
heating to 250 C for 30 min.
• They can be removed from most fluid materials by adsorption
using special materials.
• Toxins and Invasive Enzymes
• According to difference on the chemical composition,
toxicity, bioactivity, antigenicity and so on, bacterial toxins
can be divided into exotoxin (polypeptide or protein) and
endotoxin (LPS).
• Invasive enzymes secreted by bacteria can help bacteria
to invade host. So they contributed to the pathogenesis of
bacteria.
• Antibiotics
• The substance produced by many fungi and a
small number of bacteria that selectively kill or
inhibit other organisms.
• Vitamins
• A few bacteria produce various vitamins, e.g.
some certain bacteria growing in the intestine
produce vitamin K. This action is thought to be
beneficial to the host.
• Bacteriocins
• Substances produced by specific strains of bacteria
that are lethal against other strains of the same or
related species.
• They are protein or lipopolysaccharide-protein
complexes.
• pigments
• Produced by a small number of bacteria with
characteristic colors.
I) water soluble
Pseudomonas aeruginosa can produce a green water soluble
pigment, so that the color is distributed throughout the culture.
II) liposoluble
Staphylococcus aureus can produce a golden yellow liposoluble
pigment and this color only show in their colonies.
• Help to identify some of bacteria.
Pseudomonas aeruginosa produces a
blue-green pigment, which diffuses
into the medium giving the plate a
characteristic color.
Staphylococcus aureus produces a
golden yellow pigment and give the
colonies this color.
The medical significance of these products
Products
Medical significance
pyrogen
toxin
Pathogenicity of bacteria
Invasive enzyme
Antibiotic
Vitamin
Treatment of infectious
diseases
Bacteriocin
Identification of bacteria
pigment
Antibiotics
and
Antibiotic Resistance
Antibiotics
Antibiotics are powerful medicines that fight
bacterial infection
Literal translation
• anti – against
• biotic – living things
How antibiotics work
Antibiotics can be either
• Broad Spectrum
– Kill a wide range of bacteria e.g. Penicillin
• Narrow Spectrum
– Kill a specific type or group of bacteria e.g.
Isoniazid, Rifampicintablets
Antibiotics work in one of two ways
• Bactericidal
– Kills the bacteria
• Bacteriostatic
– Prevents the bacteria from dividing
Miracle Cure?
– Before the 1930s there were no treatments for
bacterial infections
– Following the discovery of penicillin industry
started searching for more antibiotics in nature
– Streptomycin was the first drug to have an effect on
tuberculosis – a condition previously untreatable
– Surgeons could attempt more dangerous operations
Miracle Cure?
Overuse of antibiotics can damage our normal/good bacteria.
– Many antibiotics prescribed by the
doctor are broad spectrum
– These kill the body’s good bacteria
as well as the bad
– With the good bacteria gone there
is more room for bad microbes to
invade!
Miracle Cure?
Antibiotics resistance

Many bacteria have developed the ability to become
resistant to antibiotics.

These bacteria are now a major threat in our hospitals.
Antibiotic resistant bacteria
aureus (MRSA)
include Methicillin Resistant Staphylococcus
Antibiotic Resistance
The Causes
– Overuse
• Antibiotics used to treat infections when they are
not needed or not effective i.e. for the flu
– Misuse
• Not completing a prescribed course
• Using antibiotics not prescribed for you
Bacterialcidal Mechanism
Cell Membrane
Cell Wall
Mitochondria
DNA
Ribosomal
Complex
Bacterialcidal Mechanism
• Damaging or inhibiting synthesis of the bacterial cell
wall (penicillins, cephalosporins, monobactams,
carbapenems, bacitracin, vancomycin, cycloserine, fosfomycin)
• Damaging or inhibiting synthesis of the cell
membrane (polymyxins)
Bacterialcidal Mechanism
• Metabolizing or inhibiting DNA synthesis of nucleic acids
(rifampin, nitrofurantoins, nitromidazoles)
• Modifying ribosomal energy metabolism (sulfonamides,
trimethroprim, dapsone, isoniazid)
• Inhibiting ribosomal protein biosynthesis (aminoglycosides,
tetracyclines, chloramphenicol, erythromycin, clindamycin, spectinomycin,
mupirocin, fusidic acid)
Acquired Bacterial Resistance
Virus
Plasmid
Acquired Bacterial Resistance
• Receiving a plasmid bearing a resistance gene from
another bacterium directly
• Receiving a resistance gene from other bacterium by
viral transfection
• Chromosomal mutation
• DNA scavenged from dead bacteria
Mechanisms of resistance
Imipenem resistant
Pseudomonas
aeruginosae
Streptococcus
pneumoniae
resistance to
penicillins
MRSA
penicillin
binding protein
PBP2A
Tetracycline
Penicillins,
Cephalosporins
Mechanisms of resistance
• Antibiotic modification: some bacteria have enzymes that
cleave or modify antibiotics: e.g. β-lactamase inactivates
penicillin
• Denied access: membrane becomes impermeable for
antibiotic: e.g. imipenem
• Pumping out the antibiotic faster than it gets in: e.g.
tetracyclines
• Altered target site: antibiotic cannot bind to its intended
target because the target itself has been modified
• Production of alternative target (typically enzyme): e.g.
Alternative penicillin binding protein (PBP2a) in MRSA
Loss of Antibiotic Resistance
• Antibiotic resistant bacteria are at a selective
disadvantage - they must expend energy and
resources to manufacture proteins that confer
resistance
• The prevalence of resistant bacteria declines after
antibiotics are withdrawn
How antibiotic resistance can be prevented
– Antibiotics should be the last line of defence NOT
the first
• Most common infections will get better by themselves
through time, bed rest, liquid intake and healthy living.
– Only take antibiotics prescribed by a doctor
– If prescribed antibiotics, finish the course.
– Do not use other peoples or leftover antibiotics
• they be specific for some other infection
Summary
1) Definitions: pyrogen, antibiotic and acteriocin
2) Bacteria growth curve, especially the characteristics and application of log
phase and maximum stationary phase.
3) The medically important antibiotic products of bacteria
4) The requirements of bacterial growth
5) The mode of bacterial reproduction
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