Week 6 - Chemoorganotrophy and fermentation Flashcards
1
Q
Chemoorganotrophs
A
energy, electrons, and carbon all obtained by breaking down organic compounds
2
Q
Chemoorganotrophs found wherever
A
there is a supply of organic compouds
tend to predominate wherever
a. there is a supply of organic compounds plus oxygen
b. there is a supply of organic compounds and no light (whether oxygen is present or not)
3
Q
Examples of chemoorganotroph habitats
A
• soil (if light tends to be aerobic, in anaerobic is usually also dark)
• ocean sediments
• our bodies
eg on the skin (plenty of organic compounds, oxygen)
in our guts (may be anaerobic, but also dark)
4
Q
All significant pathogens are
A
chemoorganotrophs
5
Q
Glycolysis
the Embden-Meyerhof pathway
A
Glucose + 2ADP + 2 Pi + 2NAD+
–>
2 pyruvate + 2ATP + 2 NADH + 2H+
6
Q
Glycolysis is common to
A
respiratory and fermentative metabolism
7
Q
Glycolysis overall production
A
2 ATP and 2 NADH per glucose molecule
8
Q
Significant further oxidation of pyruvate also requires
A
an electron acceptor - eg oxygen
• in the absence of an electron acceptor == fermentation
9
Q
Fermentation is basically a way of
A
removing excess carbon and electrons in the cell
10
Q
Because of the small amount of energy released by glycolysis
A
not all the carbon and electrons can be used for anabolic reactions
11
Q
Examples of fermentation
A
lactic fermentation
• Streptococcus
• Lactobacillus
ethanolic fermentation
• yeasts
• some bacteria
12
Q
Lactic fermentation
A
pyruvate –> lactate
NADH –> NAD+
13
Q
Ethanolic fermentation
A
pyruvate –> acetaldehyde
gives off CO2
acetaldehyde –> ethanol
NADH –> NAD+
14
Q
Other fermentation pathways lead to production of
A
Propionic acid
• Propionibacterium
Butanediol
• Enterobacter
Butyric acid etc
• Clostridium
Acetic acid + formic acid + ethanol
• many enteric bacteria
15
Q
Many of the classic tests for bacteria are based on
A
fermentation products
16
Q
Fermentation is NOT THE SAME AS
A
anaerobic respiration
17
Q
In aerobic respiration
A
pyruvate is oxidized further
eg the tricarboxylic acid cycle (aka citric acid or Krebs cycle)
18
Q
Tricarboxylic acid cycle
aka citric acid or Krebs cycle
A
pyruvate is oxidized further
Pyruvate + 4NAD+ + FAD
–>
3CO2 + 4NADH + FADH
1 ATP formed
19
Q
After the tricarboxylic acid cycle
A
the NADH and FADH are re-oxidized by the respiratory electron transport chain - located in the plasma membranes of aerobically growing bacteria
eg
2 NADH + O2 + 2H+
–>
2NAD+ + 2 H2O
20
Q
For every NADH oxidized
A
up to 3 molecules of ATP formed
21
Q
For every FADH oxidized
A
up to 2 molecules of ATP formed
22
Q
For every glucose molecule oxidized
A
38 molecules of ATP form
2 ATP/glucose from fermentatin
glycolysis makes pyruvate
pyruvate through fermentation OR
pyruvate oxidized futher (aerobic respiration - tricarboxylic acid cycle / Krebs / citric acid cycle)
23
Q
Examples of chemoorganotrophs
A
- E. coli
- Pseudomonas sp.
- Streptococcus pyogenes
- Mycobacterium tuberculosis
- Bdellovibrio
24
Q
E. coli is
A
a gram negative eubacterium
• motile - flagella enable it to swim toward food sources
• able to live on a range of sugars etc - aerobic metabolism on the presence of oxygen, but switches to fermentation when oxygen is scarce
25
E. coli's natural habitat is
in the colon of animals
• an enterobacterium
• also found in sewage-polluted water, contaminated food, etc
26
E. coli's activities in the colon
• converts food nutrients into bacterial biomass
• uses oxygen - maintains anaerobic conditions in the lower colon
• produces vitamin K
• may cause disease (occasionally fatal)
- diarrhoea
• septicaemia
• urinary tract infection
27
Pathogenic strains of E. coli
produce toxins and often have fimbrae
• eg strains responsible for traveller's diarrhea have genes coding for 2 polypeptide toxins, and genes coding for adhesive fimbrae that allow colonization of the small intestine
28
E. coli -->
diarrhoea
29
Infectious diarrhoea
* 3-5 billion episodes yearly
* major cause of worldwide morbidity and mortality
* 5 million deaths yearly - 80% are <1 year old
* major cause of school/work absenteeism
* major economic burden, especially in developing countries
* bacteria cause 80% of foodborne diarrhoea
30
Infectious diarrhoea
| organisms
Bacteria
• E. coli, Salmonella, Campylobacter, Vibrio, Yersinia, Clostridium difficle, S. aureus, B. cereus, C. botulinum
Viruses
• Norovirus, Rotavirus, CMV
Parasites
• Giardia, Amoeba, Ascaris
31
These organisms cause diarrhoea through a wide variety of mechanisms
```
pathophysiology
• osmotic
• secretory
• exudation
• abnormal motility
```
32
Daily intake and endogenous secretions are
efficiently absorbed by the gastrointestinal tract
33
Osmotic diarrhoea
increased amounts of poorly absorbed, osmotic active solutes in gut lumen
• interferes with absorption of water
• solutes are ingested (FASTING STOPS DIARRHOEA)
- magnesium sulfate, citrate, or magnesium containing antacids
- sorbitol
- malabsorption of food (variety of infectious organisms, particularly viruses but including E. coli)
34
Secretory diarrhoea
excess excretion of electrolytes and water across mucosal surface
• usually coupled with inhibition of absorption
• clinical features
- stool is very watery
- stool volume large
FASTING DOES NOT STOP DIARRHOEA
35
Diarrhoea associated with active secretory processes (secretory diarrhoea) can be caused by stimuli from 3 sources
* mucosal/submucosal stimuli
* blood hormone stimuli
* liminal stimuli
36
```
Secreotry diarrhoea
(organisms)
```
```
bacterial or viral enterotoxins
• Vibrio cholerae
• Noncholeraic vibrios
• enterotoxigenic E. coli
• B. cereus
• S. aureus
• others: Rotavirus, Norovirus
```
37
Exudative diarrhoea
* intestinal or colonic inflamed and ulcerated
| * the extent and location of bowel involved determines severity
38
Common syndromes of infectious diarrhoea
```
• food poisoning
• acute watery diarrhoea
- travelers diarrhoea
- epidemics
• acute bloody diarrhoea
- dysentery
```
39
Special circumstances
* outbreaks/food poisoning
* overseas travel
* immunocompromised host
* raw seafood ingestion
* antibiotic usage
40
E. coli O157:H7
1982 - first recognized as a pathogen
1985 - associated with hemolytic uremic syndrome
1990 - outbreak from drinking water
1991 - outbreak from apple cider
1993 - multi-state outbreak from fast food hamburgers
1995 - outbreak from fresh produce
1996 - outbreak in Japan, multi-state outbreak from unpasteurized apple juice
41
E. coli 0157
petting zoos
42
E. coli 0157 case history
* onset and duration of diarrhoea
* timing of exposure to potential pathogens: travel, ingestion history, environment, recent medications (antibiotics), age
* character of stool: volume, presence of blood, mucus, or pus
* associated symptoms and signs: abdominal pain, fever, vomiting, dehydration
43
E. coli 0157
| physical examination
* vital signs: fever, tachycardia
* abdominal tenderness or pain
* signs of dehydration
* blood, mucus, or pus in stool
44
Type 1
separate hard lumps, like nuts (hard to pass)
45
Type 2
sausage-shaped but lumpy
46
Type 3
like a sausage but with cracks on the surface
47
Type 4
like a sausage or snake, smooth and soft
48
Type 5
soft blobs with clear-cut edges
49
Type 6
fluffy pieces with ragged edges, a mushy stool
50
Type 7
watery, no solid pieces, entirely liquid
51
E. coli cow ingestion cycle
1. ingestion of pathogenic E. coli
2. fecal excretion + contamination of environment
345. contamination of food + transmission animal to person + transmission person to person
6. colonization of the large intestine + intimate attachment to epithelial cells
7. transport of toxin to circulation
8. bloody diarrhoea + kidney failure
52
Classification
chromogenic media
53
E. coli O157 chromogenic media classification
* sorbitol-fermenting bacteria such as most E. coli appear as pink to red colonies
* on Rainbow Agar O157 or R&F E. coli O157 should appear as black to blue-black colonies
54
Serotyping
pathgenic E. coli may also be differentiated by serotyping based on antigenic differences in
• the O antigen of the LPS
• in the flagellar or H antigens
• and the fimbral or F antigens
55
EC virotype classification
each class falls within a serological subgroup and manifests distinct features in pathogenesis
• ETEC: enterotoxigenic
• EHEC/STEC: enterohaemorrhagic / Shiga toxin
• EIEC: enterinvasive
• EAEC/AEEC: enteoaggrigative / attach effacing
• EPEC: enteropathogic
56
A pathogenic E. coli within each pathotype may be further classified as
virotypes, based on the virulence gens that they possess
57
Virotype
combination of virulence genes
58
Important virulence factors encoded by these genes include
* fimbrial adhesins
* enterotoxins
* cytotoxins
* capsule
* lipopolysacccharide (LPS)
59
Virotype/pathotype
| ETEC
* watery diarrhoea, travelers diarrhoea
| * complications rare
60
Virotype/pathotype
EHEC or STEC
(Shiga toxin 1 and 2)
* bloody diarrhoea
| * haemolytic uremic syndrome HUS (mostly O157:H7)
61
Virotype/pathotype
| EIEC
* bloody diarrhoea, dysentery
| * complications rare
62
Virotype/pathotype
| EAEC
* watery diarrhoea or bloody diarrhea, mainly in children
| * may be protracted
63
Virotype/pathotype
| EPEC or AEEC
* watery, sometimes bloody diarrhoea
| * leading cause of infant diarrhoea
64
ET (toxigenic) E. coli produces LT and ST
* produces a heat-labile enterotoxin (LT) similar to the cholera toxin (Ctx)
* ETEC can also produce a heat stabile toxin (ST) - this causes an increase in cyclic GMP in host cell. ST binds and activates guanylate cyclase on the membranes of host cels. this leads to secretion of fluids and electrolytes resulting in diarrhoea
* ETEC adhesins are fimbral adhesins - adhere to receptors on the small intestine
* symptoms ETEC infections include diarrhoea without fever
* the bacteria colonize the GI tract by means of a fimbral adhesin, and are noninvasive, but produce either LT or ST toxin
65
ETEC process
1. ingestion of pathogenic E. coli
2. colonization of the jejunum and ileium
3. water and electrolytes
4. diarrhea, weight loss, death
66
ETEC
surface doesn't change much, adheres to gut
| • delivery of LT or ST enterotoxins
67
EI (invasive) EC
* penetrate and multiply within epithelial cells of the colon causing widespread cell destruction
* the clinical syndrome is identical to Shigella dysentery and includes a dysentery-like diarrhoea with fever
* EIEC apparently lack fimbral adhesins but do prossess a specific adhesin that, as in Shigella, is thought to be an outer membrane protein
* they do not produce LT or ST toxin
68
EIEC
```
surface of host destroyed by toxins, sink and absorbed, horizontal transfer
• invasion
• phagosomal rupture
• intracellular movement
• lateral spread to adjacent cell
```
69
EP (pathogenic) EC
• pathogenesis of EPEC involves
- plasmid-encoded protein referred to as EPEC adherence factor (EAF) that enables localized adherence of bacterial to intestinal cells and
- a non fimbral adhesin designated intimin, whis is an outer membrane protein that mediates the final stages of ahderence
• they do not produce ST or LT toxins
• EPEC strains adhere to the intestinal mucosa, they produce changes in the ultrastructure of the cells
• they cause inflammatory response
• the diarrhoea and other symptoms of EPEC infections are caused by invasion NOT toxins
70
EPEC (picture)
adhere (fimbrae), toxins kill flagella, inject agent into cell
• initial adherence via BFP
• intimate attachment
• actin condensation and microvillous effacement
71
EAEC
* strains attach to tissue culture cells in an aggregative manner
* these strains are associated with persistent diarrhoea in young children
* they resemble ETEC strains in that the bacteria adhere to the intestinal mucosa and cause non-bloody diarrhoea without invading or causing inflammation
* the organisms produce an enterotoxin. a heat-labile plasmid-encoded toxin has been isolated from these strains, called the EAST (EnteroAggregative ST) toxin
* they also produce a haemolysin related to the haemolysin prooduced by E. coli strains involved in urinary tract infections
* the role of the toxin and the haemolysin in virulence has not been proven, the significance of EAEC strains in humans is controversial
72
EAEC
knocked out all flagella, sits on surface, toxin goes into cell
• aggregative adherence
• mucus biofilm
• delivery of cytotoxin
73
STEC / EH (haemohorrhagic) EC
* EHEC causes bloody diarrhoea, which can progress to the potentially fatal haemolytic uremic syndrome (HUS)
* EHEC are characterized by the production of verotoxin or Shiga toxins (Stx). Although Stx1 and Stx2 are most often implicated in human illness, several variants of Stx2 exist
* O157:H7 is the prototypic EHEC and most often implicated in illness worldwide
* the infectious dose for O157:H7 is 10-100 cells
* EHEC infections are mostly food or water borne
74
STEC Edema disease
1. ingestion of pathogenic E. coli
2. colonization of the jejunum and ileum
3. transport of toxin to circulation
4. edema, ataxia, death
75
EHEC
toxin through cell to blood stream --> kills you
• Shiga toxin kills flagella, shiga toxin through cell --> blood
• intimate attachment of bacteria
• actin condensation and microvillous effacement
• delivery of shiga toxin
76
EcL approach to identification of pathogenic E. coli
| Sample analysis
* sample
* enrichment (over night)
* multiplex PCR
* rapid evaluation of the virulence potential of the sample (pathotype)
77
EcL approach to identification of pathogenic E. coli
| Isolate analysis
* E. coli isolates
* agar plates
* virotyping (multiplex PCR or colony hybridization)
* serotyping
* pulse typing (PFGE)
* identification and virotyping of the isolate
78
Alcohol gels are NOT effective against E. coli in the presence of
feces
79
Pseudomonas - versatile, ecologically important
* Pseudomonas species are aerobic
* gram negative rods
* motile because of flagellae
* significant in the environment and also the cause of plant and animal diseases
* opportunistic infections
* versatile metabolism - able to use many different organic compounds as carbon and energy sources
80
Pseudomonas - versatile metabolism
able to use many different organic compounds as carbon and energy sources
81
Example of versatility
| Pseudomonas aeroginosa
* primarily a soil microbe
| * is also a serious opportunistic pathogen - infects wounds, abcesses, burns, etc
82
Streptococcus pyogenes
* gram positive
* forms chains
* can grow aerobically or anaerobically
* responsible for numerous infections
83
Erysipelas
* infection of the dermal layer of the skin with Streptococcus pyogenes
* produces toxins and breaks down host tissue
84
Respiratory diseases
* infections of the upper respiratory system are the most common type of infection
* pathogens that enter the respiratory system can infect other parts of the body
85
Tuberculosis
• bacterial disease of the lower respiratory system
86
Tuberculosis
| pathogenesis
M. tuberculosis can remain viable for long periods in aerosol drops
• 3 types of tuberculosis
- primary tuberculosis - initial case of tuberculosis disease
- secondary tuberculosis - reestablished tuberculosis
- disseminated tuberculosis - tuberculosis involving multiple systems
87
Primary tuberculosis
initial case of tuberculosis disease
88
Secondary tuberculosis
reestablished tuberculosis
89
Disseminated tuberculosis
tuberculosis involving multiple systems
90
Tuberculosis epidemiology
* immunocompromised individuals are most at risk
| * tuberculosis is the leading killer of IHV+ individuals
91
The pathogenesis of tuberculosis
1. tubercle bacilli that reach the alveoli of the lung are ingested by macrophages, but often some survive. Infection is present, but no symptoms of disease
2. Tubercle bacilli multiplying in macrophages cause a chemotactic response that brings additional macrophages and other defensive cells to the area. These form a surrounding layer and, in turn, an early tubercle. Most of the surrounding macrophages are not successful in destroying bacteria but release enzymes and cytokines that cause a lung-damaging inflammation
3. after a few weeks, disease symptoms appear as many of the macrophages die, releasing tubercle bacilli and forming a caseous center in the tubercle. the aerobic tubercle bacilli do not grow well in this location. However, many remain dormant (latent TB) and serve as a basis for later reactivation of the disease. The disease may be arrested at this stage, and the lesions become calcified.
4. In some individuals, disease symptoms appear as a mature tubercle is formed. The disease progresses as the caseous center enlarges in the process called liquefaction. The caseous center now enlarges and forms an air-filled tuberculous cavity in which the aerobic bacilli multiply outside the macrophages
5. Liquefaction continues until the tubercle ruptures, allowing bacilli to spill into a bronchiole and thus be disseminated throughout the lungs and then to the circulatory and lymphatic systems
92
Mycobacterium tuberculosis VNTR typing
* the ability to link related cases of tuberculosis (TB) infection and exclude unrelated cases in important for epidemiologiology
* various methods can be used to track spread of infection and DNA typing of M. tuberculosis cases has recently focused on Variable Number Tandem Repeat (VNTR) DNA sequences as markers in the genome that show variation between unrelated strains
* VNTR- PCR amplification is a method that involves amplifying the DNA using PCR primers in the sequences flanking this region to detectable levels. The number of tandem repeat units can be determined from the size of the PCR product using various methods (including automated fragment analysis and gel electrophoresis)
93
Various methods can be used to track spread of infection and DNA typing of M. tuberculosis cases has recently focused on
Variable Number Tandem Repeat (VNTR) DNA sequences as markers in the genome that show variation between unrelated strains
94
VNTR-PCR amplification is a method that involves
* amplifying the DNA using PCR primers in the sequences flanking this region to detectable levels
* the number of tandem repeat units can be determined from the size of the PCR product using various methods (including automated fragment analysis and gel electrophoresis)
95
TB
* about 1/3 of the world's population is infected with Mycobacterium tuberculosis
* it is estimated that currently there are about 10 million new cases of TB every year with 3 million deaths occurring world-wide
* death from TB comprise 25% of all avoidable deaths in developing countries
* in India TB has the greatest incidence
96
About 1/3 of the world's population is infected with
Mycobacterium tuberculosis
97
It's estimated that currently there are about
10 million new cases of TB every year with 3 million deaths occurring world-wide
98
death from TB comprise
25% of all avoidable deaths in developing countries
99
Benefits of TB genotyping
When combined with epidemiologic data
Confirm epidemiologic links
• outbreaks detected earlier - controlled more rapidly
• detect unsuspected transmission
• transmission between patients living in different jurisdictions are detected more readily
• detect or confirm false positive cultures
• able to monitor progress toward the elimination of TB transmission
100
The biggest bacterial killer of all Homo sapiens
Mycobacterium tuberculosis
| 1.5-3 million deaths per year
101
Mycobacterium tuberculosis
| x deaths per year
1.5-3 million deaths per year
102
Mycobacterium tuberculosis
* 1-5 - 3 million deaths per year
* gram-positive
* aerobic eubacterium
* first isolated and identified as the cause of tuberculosis by Robert Koch (1880s) - Nobel Prize 1905
103
M. tuberculosis invades lung tissue
• cells often survive being engulfed by macrophages
and can grow and divide inside macrophage cells
• destruction of lung tissue can eventually lead to death
• infection spread from individual to individual by coughing (infection can also be contracted from contaminated milk)
• treatment with a cocktail of chemotherapeutic agents and antibiotics
• but resistant strains are an increasing problem in some regions - high rate of spontaneous mutations
• genome now sequenced - hope for development of new chemotherapeutic agents
104
M. tuberculosis - infection spread from individual to individual by
coughing
| • infection can also be contracted from contaminated milk
105
M. tuberculosis - resistant strains
an increasing problem in some regions
| • high rate of spontaneous mutation
106
Bdellovibrio bacteriovorus
* gram-negative
* aerobic
* curved rod
* with a flagellum
* small, highly motile cells
* widespread in soil and water
* bacterial predator - enters the periplasm of other bacteria, eats them from the inside, reproduces in the carcass
107
Enters the periplasm of other bacteria, eats them from the inside, reproduces in the carcass
Bdellovibrio bacteriovorus
