Lecture 9- Gram positive bacteria – Topic 4A The endospore formers Bacillus

Question 1

The Firmicutes Low GC Gram-positive bacteria

Answer 1

Non Spore forming bacteria

The lactic acid bacteria
Streptococcus
Lactococcus
Lactobacillus
Staphylococcus

Listeria
Mycoplasma

The spore forming bacteria
Clostridium
Bacillus

Question 2

Actinobacteria –
the high GC Gram positive bacteria

Answer 2

Streptomyces
Nocardia
Corynebacteria
Mycobacteria

Question 3

The endospore forming bacteria

Answer 3

these bacteria create a specialised cell called an endospore and these are produced when conditions for growth become unsuitable such as nutrient limitation and so the cell goes into a spore form which is basically a dormant version of the bacteria that is highly reistant to different extreme stresses like heat, UV radiation or toxic chemicals and pressure so is a way for bacteria to protect themselves when conditions for growth are not suitable
typical habitat for endospore forming bacteri is soil

Main genera: Bacillus - aerobic or facultative anaerobes
Clostridium - anaerobic

*Form a distinctive type of dormant cell - the endospore.
*Usually formed when a population reaches conditions of nutrient
limitation.
*Highly resistant to heat, uv, toxic chemicals, ionising radiation.
*Heat resistance is used for selection.
*Typical G + cell wall, but stain variably.
*Typical habitat is soil.

Question 4

The sporulation
cycle of Bacillus

Answer 4

germination occurs when conditions are favourable for growth

Question 5

Spore Structure

Answer 5

Exosporium – thin delicate layer of mostly protein

Spore coat(s) – multiple layers of spore specific proteins

Cortex – loosely packed peptidoglycan

Spore protoplast or core – normal cell wall
plasma membrane
cytoplasm and nucleoid

Question 6

Properties of the Core- important fcats

Answer 6

• Dipicolinate (dipicolinic acid)– protects DNA- 10% of mass of spores is dipicolinic acid; very specific to these spores; this compound is not commmonly found elsewhere
• Dehydrated – only 10-30% of the water content of the vegetative cell
  –gives heat and stress resistance
• pH is more acidic than vegetative cell
• Abundant small acid-soluble spore proteins, SASPs – give resistance
  to dry heat, dessication, UV – protecting DNA Carbon source during
  outgrowth

Question 7

Properties of the Core

Answer 7

they are a dormant cell in a state of suspended animation
* Low metabolic activity (low O2 uptake)
* No macromolecular synthesis
* Low enzymatic activity
* Low or absent mRNA synthesis

Question 8

Germination

Answer 8

Spores will remain dormant for decades, even if placed in optimal conditions.
State of dormancy can be broken by a variety of treatments - activation.
heat shock. eg. several hours at 65C
storage at low temperature (4C)
When activated spores are placed in favourable conditions - germination

1. Activation of cell
2. Germination
3. Outgrowth from the spore

Question 9

How old are the oldest living cells in Nature?

Answer 9

The oldest living cells in nature are generally considered to be microorganisms, particularly bacteria and archaea, which can live for extremely long periods of time. While it is difficult to pinpoint the exact age of individual cells, some of the oldest living cells include:

1. Microbial Life in Dormant States
   Sporulation: Certain bacteria, like Bacillus and Clostridium species, can form endospores that allow them to survive in extreme conditions for potentially millions of years. Endospores can remain dormant and viable for extraordinarily long periods until they find favorable conditions for growth.
   Permafrost Microbes: Microbes found in permafrost (frozen soil) have been shown to survive for tens of thousands to possibly millions of years. For example, bacteria isolated from the Siberian permafrost have been estimated to be up to 600,000 years old.
   Salt Crystals (Halophiles): Halophilic bacteria and archaea have been found in salt crystals that date back to the Permian period, around 250 million years ago. These microorganisms remained viable in a dormant state, protected by the crystal environment, and can be revived when the conditions are suitable.
2. Deep-Sea and Subsurface Microbes
   Subsurface Bacteria and Archaea: Microbes living deep within Earth’s crust or in oceanic sediments can have incredibly slow metabolic rates, allowing them to live for extremely long periods. These microorganisms can remain active for millions of years, sustaining themselves on minimal energy in nutrient-poor environments.
   Ancient Seafloor Sediments: Some microbes have been found in ancient marine sediments that are estimated to be up to 100 million years old. These organisms survive with very low metabolic activity, using trace amounts of nutrients in the sediment.
3. Stromatolites
   Stromatolites are layered structures formed by the growth of cyanobacteria and other microorganisms. While the structures themselves are not individual cells, cyanobacteria within modern stromatolites are thought to be descendants of some of the oldest forms of life on Earth, dating back 3.5 billion years.
   These ancient microbes have evolved over time, but the cellular lineage of cyanobacteria is one of the oldest on the planet, making them among the earliest forms of life.
4. Methuselah Cells
   While not a specific organism, the concept of Methuselah cells refers to microbial cells that can remain in suspended animation or extremely slow metabolic states for extensive periods. These cells can potentially remain alive for millions of years under the right conditions.
   Key Factors for Longevity of Cells
   Dormancy and Low Metabolism: Cells that can enter a dormant state, reduce their metabolic activity, or form protective structures (like spores) are more likely to survive for long periods.
   Extreme Environments: Cells found in extreme conditions (cold temperatures, deep-sea sediments, or highly saline environments) are often protected from damage and have reduced metabolic needs, contributing to their longevity.
   Protection from DNA Damage: Dormant cells have mechanisms to protect their genetic material from damage caused by radiation, desiccation, or other environmental stresses, increasing their chances of long-term survival.
   Conclusion
   The oldest living cells are primarily microorganisms that have adapted to survive in extreme environments or enter dormant states to withstand the test of time. Some microbial life has been revived after being dormant for millions of years, making these ancient cells the longest-lived known life forms on Earth.

Question 10

Longevity of spores.

Answer 10

Spores are extremely stable and can survive for long periods. Previously
the best documented is 70 years however recently (1995!) spores have
supposedly been revived from the gut of a bee preserved in fossil amber
25-40 million years old! Sequenced rRNA genes - related to extant
Bacillus sphaericus.

New record is (maybe!!!) the halophilic spore forming bacterium isolated
from salt crystals over 250 million years old.

Question 11

genus Bacillus

Answer 11

*Aerobic or facultative anaerobes.
*Variable Gram stain.
*Habitat: mainly soil organisms, although some parasites
and pathogens.

Bacillus anthracis produces mucoid colonies because they have capsules

Question 12

Bacillus anthracis

Answer 12

Agent of anthrax. Disease of sheep, goats & cattle, that is
also transmissable to humans. Isolated by Koch in 1877 - first
bacterial pathogen. Survive in soil for 30 years or more.

bacillus has a classic bacillus shaped block end it is a large bacteria, in an elongated bacillus form

diagnostic feature in endospore forming bacteria is where the spore forms and here it forms in the centre of the cell and refractile; spores do not stain very well

Question 13

Cutaneous Anthrax

Answer 13

-Most common form (2000ish cases per year)
-common in ungulates (hooved animals)
*Animal workers mostly- can be infected if they have cuts on their hands that get contaminated by spores of bacillus anthracis that then germinate and start replicating to release their exotoxins to create a necrotic lesion that will then allow the bacilli to become systemic and start spreading throughout the body
*Spores germinate in skin abrasions
*Skin ulcer – black eschar – can become systemic
*Untreated 20% mortality, responds well to antibiotic

Question 14

Gastrointestinal Anthrax

Answer 14

*ingestion of undercooked contaminated meat
*2 types - oral-pharyngeal (most rare) and abdominal (rare)

Abdominal
*Spores germinate in lower gastrointestinal tract
*primary intestinal lesion forms
*Symptoms - nausea, severe abdominal pain, vomiting, bloody diarrhea
*Intestinal perforation or anthrax toxemia are the usual causes of death
*Mortality very high as perforation of the gut means lots of bacteria will start leaking into the body cavity and causing lots of infections leading to toxic shock, but even if you don’t get perforation in the gut the effects of the toxin will be felt around the body so death is likely

Question 15

Pulmonary anthrax

Answer 15

*Inhalation of spores that then land on the alveolar surfaces in your lungs
*Some spores are mopped up by
macrophages which then move around the body and travel to lymph nodes
*Others are trafficked to the
draining lymph nodes
*Germination in the lymph nodes
may take up to 60 days
*Anthrax bacilli replicate in the lymph nodes forming a necrotic lesion and disease immediately follows as bacteria starts spreading systemically. the Capsule inhibits phagocytosis.
*Hemorrhage, edema, and necrosis are the results of bacterial exotoxins released during replication
*Symptoms- flu-like with fever, myalgia, cough, headache, vomiting, chills,
abdominal pain, and chest pain. Cyanosis and hypotension result in death
-regardless of the type of anthrax the symptoms in common will be haemorrhaging blood vessels, oedema and necrosis of tissue
*80% mortality in 2 to 4 days whether antibiotics given or not. Inhalational
anthrax is 99% lethal in unvaccinated individuals.
*Biological warfare

Question 16

Virulence of Bacillus anthracis

Answer 16

Capsule made of poly-D-glutamate polypeptide, this capsule protects the bacteria from phagocytosis and from being destroyed by the macrophage. produces Smooth mucoid colonies.
capsule genes are encoded in pX02 plasmid. Resists phagocytosis and complement system.

Toxin encoded on plasmid pX01. 3 parts
all these parts are needed for the bacteria to be virulent

• PA (binding domain)
• EF (Edema factor)
• LF (Lethal factor)

Question 17

Bacillus anthracis diagnosis

Answer 17

Diagnosis
G+ rod. Central (non-staining spore) occurs in soil and in culture, but not
in clinical samples. Sometimes occur in chains. Identified in blood, skin lesions, or
respiratory secretions or by measuring specific antibodies in the blood. Extreme
caution is required when handling samples.
the bacteria will not be in spore form during infection

Treatment - penicillin, doxycycline, and fluoroquinolones (such as
ciprofloxacin). To be effective, treatment should be initiated early.

Prevention
Pasteur demonstrated efficacy of vaccine. Attenuated vaccine possesses
some toxicity. Used for occupational exposure.
Control also relies on sterilisation of wool etc. from areas where anthrax is endemic.
There are presently three vaccines:
the Georgian/Russian, the UK, and the US vaccines.
All derived from an avirulent strain of B. anthracis - lacks the plasmid pX02 which
encodes the protecive coating of the bacteria. The Russian vaccine is inoculation
with live spores - high of side effects.
UK and US is dead cell free preparation of bacterial filtrates

Question 18

Bacillus cereus

Answer 18

One of the most abundant aerobic sporeformers found in soil.
Distinctive loosely spreading (Medusa) colonies, superficially resembling
fungi.

Question 19

Bacillus cereus – food poisoning

Answer 19

for the emetic version, the heat spores may be activated by the boiling which wakes them up and causes them to germinate and the toxin produced is heat stable so it does not matter if you reheat the food after

the diarrheal form is caused by live bacillus cereus, this type of poisoning is caused by a different toxin, the toxin is produced by the bacteria replicating in the gut

Question 20

Gram positive
bacteria
Topic 4B
The endospore formers
Clostridium

Answer 20

these are all anaerobes
very good at decomposing proteins

Question 21

Clostridia - Fermentation

Answer 21

• Clostridia lack a cytochrome system and a mechanism for
  electron transport phosphorylation – therefore they obtain
  ATP only via substrate-level phosphorylation.
• The wide variety of fermentable substrates used and
  mechanisms to derive energy from these can be used to
  group the clostridia.
• Some ferment sugars producing end products which may
  include butyric acid, butanol and acetone (once industrially
  important).

Question 22

Pathogenic Clostridia

Answer 22

Natural habitat is soil and the intestinal tract of man and
animals. Invasive capability of pathogenic clostridia varies
considerably.

At one end the organisms remains at the site of inoculation
(tetanus) or may not even enter tissue at all (botulism)
whereas at the other extreme the organisms elaborates
destructive enzymes that allow progressive invasion of
necrotic tissue.

Toxins are amongst the most powerful poisons known to
man - 1 million times more toxic than rattlesnake poison.

Question 23

C. Botulinum - Botulism

Answer 23

*C. botulinum is found in soil, water and decaying vegetation.
*Fatal food poisoning that follows on ingestion of preformed toxin from growth
of organism in food. Intoxification.
*Animals are frequently affected from feeds.
*Endospores are very resistant to heat and may withstand boiling for several
minutes
*Anaerobic conditions (incompletely sterilised
canning) may germinate spores - grow into
vegetative cells and eventually produce the
deadly toxin.

causes botulism food poisoning in humans and also animals, it is an intoxication where the animal or person ingests botulinum toxin in animals it can be related to botulinum toxin-containing feeds

the spores are very resistant so it is important to understand how hot and for how long you need to heat food to produce long term sterilisation for products in canning (tinned food)

Question 24

Botulinum toxin

Answer 24

botulinum toxin comes along to block the release of acetylcholine to prevent muscle contraction- this causes flaccid paralysis and if muscles cannot be contracted this can prevent breathing and thus death

Question 25

C. botulinum -

Answer 25

*May occasionally infect wounds.
*Infant botulism can occur in newborns and up to a few months and as a result of ingestion of spores in soil and
dust - germinate in intestine (anaerobic).
*Honey that’s not been pasteurised or stored correctly that is added to formula feed found to contain spores.

Diagnosis
Clinical. ELISA for toxin in food or suspect food is injected into mice. Culture is attempted.

Prevention
Antitoxin is given to suspected cases. In contrast to
spores, toxin is heat labile therefore cooking inactivates.
can be given antiserum against botulinum toxin

Question 26

BOTOX

Answer 26

Botulinum toxin type A
injected into muscle, lasts
for several months.

Used to treat patients with
overactive muscles

Eg. cross eyes (1960s)
dystonia

Question 27

Clostridium tetani

Answer 27

Causative agent of tetanus.
First described by Hippocrates
Etiology discovered in 1884 by Carle and Rattone
Commonly found in soil and intestines of animals (humans
transiently).
>more than 300,000 cases per year

associated with human and animal intestinal carriage

causes a spastic pyralysis

Question 28

Terminal spore produces a drumstick shape

Answer 28

spore produces a drumstick shap

Question 29

Tetanus - Clostridium tetani

Answer 29

scenario
- clostridium tetani is commonly found is soil and the intestines of animals so someone could be digging rotted horse manure for their plants in their garden and accidentally the person may introduce that clostridium tetani into their body through a cut or injury; this will innoculate the bacteria from the soil and into your body at a place of necrotic tissue that has had blood supply cut off due to damage by injury, in that necrotic tissue clostridium tetani spores will germinate and start replicating and thus produce their tetanus toxin that will have its effect and cause disease

*Spores are introduced into the body through wound.
*Wounding producing necrotic (dead) cells allows anaerobic conditions to
develop and spores to germinate and formation of toxin.

2 types of tetanus
=Generalised tetanus

=*Neonatal tetanus occurs if umbilical stump becomes infected - from
soil or bindings containing spores.
*Normally due to lack of passive immunity from the mother
*High fatality rate
*>200,000 deaths each year

Question 30

Tetanus - Pathogenesis

Answer 30

in both neonatal and generalised tetanus the bacteria that germinate at the site of their spores remain very localised at the site of the infection however the toxin goes further and is taken up by peripheral nerves

• Toxin migrates along peripheral nerve axons to the central nervous system (site of action)
• Symptoms take 3-21 days to develop
• causes spastic paralysis - convulsive contractions of voluntary muscles.
• Since spasms often involve neck and jaws disease is referred to as lockjaw (teeth knocked out in the past to prevent tongue being bitten off!).
• Body may be severely contorted.
• Death results from muscular spasms affecting respiration. 50% mortality.
• Symptoms last 3-4 weeks, complete recovery not for months (if at all!)

Question 31

Tetanus toxin

Answer 31

tetanus toxin inhibits the release of glycine onto the motor neuron and so you get uncontrolled and unregulated release of acetylcholine that causes spastic paralysis of the muscle cells

contrasting with botulinum toxin which inhibited the release of acetylcholine and caused flaccid paralysis

Question 32

Tetanus - Clostridium tetani diagnosis and prevention

Answer 32

Diagnosis
Diagnosis is usually by clinical picture, although organism can be isolated -
G+, drumstick shaped due to terminal spore. Haemolytic toxin production can
be demonstrated by growth on agar plates half spread with antitoxin -
prevents haemolysis.

Prevention and treatment
Prevention is by immunity produced by vaccination with formalin-inactivated
toxin (in DPT triple vaccine). Tetanus toxoid vaccine has 100% efficacy. the vaccine uses inactivated toxins

Following injury, non-immunised persons are given human tetanus
immunoglobulins.
Even in treated people, mortality may be as high as 20 - 30%.

Question 33

C. perfringens - Gas Gangrene

Answer 33

most common bacteria to cause gangrene
clostridia are anaerobic so need to grow in anaerobic conditions and so like the anaerobic conditions of necrotic tissue
in the anaerobic conditions the bacteria is able to ferment carbohydrates as a result it produces gas so you end up with pockets of gas in the tissue; hence gas gangrene, the bacteria produces enzymes to get further through the tissue which causes more damage and necrosis

necrotic tissue can be formed after an injury in which blood and oxygen is cut off from the site of the damage during healing

Question 34

C. perfringens - Gas Gangrene has a charchteristic smell

Answer 34

you can see pockets of gas in the tissue via an X-ray

Question 35

C. perfringens – more diagnosis

Answer 35

a more definitive diagnosis can be done by doing the nagler reaction
- in such a case bacteria isolated from the wound is streaked across a plate, and half the plate is covered with an antiserum to the alpha toxin produced by the bacteria
- the alpha toxin will hydrolyse the lecithin in the egg yolk media which will result in a halo effect
however, if you have the antiserum added to the toxin you will not get any halo effect around the bacteria

antiserum is specific to the toxin only produced by the clostridium perfringens

Question 36

C. perfringens - Gas Gangrene treatment an prevention

Answer 36

this is difficult to do as the lack of blood supply to dead necrotic tissue means you cannot use antibiotics as there is no blood to take it there; the best option is usually to cut off the infected tissue and take antibiotics too

Question 37

C.difficile - Pseudomembranous Colitis

Answer 37

scenario
- someone goes into hospital for surgery and as part of the normal flora in their gut they’re carrying clostridium difficile and the clostridium difficile is expressing one of two enterotoxins, enterotoxin A or enterotoxin B and usually this will not be a problem however if a person after their surgery is given antibiotics to protect their would from being infected against something like enterococcus or staphylococcus- the antibiotic will change the microflora in their guts but will not affect the clostridium difficile which is an anaerobe as it needs specific antibiotics this means the clostrisium difficile has the chance to overgrown because it no longer competes with the other bacteria int he gut that have been wiped out by the antibiotics- that clostridium can then cause colitis and diarrhoea

pseudomembranous colitis results in plaques on the inflamed walls of the intestinal tract

Question 38

C.difficile - Colitis diagnosis and treatment

Answer 38

you will need to take a sample of the poo and take it to a lab and an ELISA test

the treatment is to change antibiotics and give antibiotics that are effective against anaerobic bacteria such as metronidazole especially

Question 39

position of the spores

Answer 39