block 8- cholera

Question 1

cholera

Answer 1

• common waterbourne disease
• Cholera is an acute diarrhoeal infection caused by ingestion of food or
  water contaminated with the bacterium Vibrio cholerae.
  -Provision of safe water and sanitation is critical in reducing the impact of
  cholera and other waterborne diseases. so is a problem in poorer countrys

Question 2

the genus vibrio

Answer 2

-Gram-negative straight or curved rods, motile
by means of a single polar flagellum
-Vibrios are aquatic organisms, they occur in both marine and fresh water
habitats. Are capable of both respiratory and fermentative metabolism
-V. cholerae, V. parahaemolyticus and V. vulnificus are pathogens of humans

Question 3

vibrio cholerae

Answer 3

-a severe
diarrheol disease caused by the bacterium Vibrio cholerae.
-The Infectious Dose- ID50 varies depending on the pH of the stomach. In healthy
volunteers 108 bacteria produces infection, after neutralization of stomach acid 104 bacteria can cause disease( basically need a very high number of the bacterium to get the disease which lowers when the stomach is neutralized)
-The small intestine is the primary site of infection. V. cholerae colonise the
epithelium without invasion or apparent damage. This is an extracellular
pathogen that does not invade host tissue.

Question 4

nomenclature of v.cholerae

Answer 4

-V. cholerae has been classified according to its O antigens. These O
antigens allow for the classification of V. cholerae strains into serovars or
serogroups. At least 155 different V. cholerae serogroups had been
identified
-Serogroups can be divided into even smaller groups of classification
known as biotypes. There are two biotypes, El Tor and classical. Biotypes
are determined based on specific biochemical properties and the
bacteriophage susceptibility of the isolate.

Question 5

principles of identification of cholerae

Answer 5

-isolates from primary culture are identified by colonial appearance,
Gram’s stain, serology (agglutination with specific antisera) and
biochemical testing.
-Thiosulfate Citrate Bile Sucrose
Agar (TCBS) is used for the
isolation and selective cultivation
of Vibrio cholerae and other
enteropahtogenic vibrios.
Growth of V. cholerae on TCBS

Question 6

clinical symptoms of cholera

Answer 6

• voluminous diarrhoea and dehydration
  -Hypovolaemic shock occurs when the volume of the circulatory system is too
  depleted to allow adequate circulation to the tissues of the body

Question 7

rehydration treatment of cholera

Answer 7

-Oral Rehydration Salts (ORS)
-Packets of Oral Rehydration Salts Distributed by WHO, UNICEF
-Dissolve in 1 L water
NaCl, KCl, NaHCO3, glucose
In severe cases a drip is needed to
start the process
An effective antibiotic can reduce the volume and duration of
diarrhoea and the period of Vibrio excretion. Tetracycline is the usual
antibiotic of choice, but resistance to it is increasing. Other antibiotics
that are effective when V. cholerae are sensitive to them include
cotrimoxazole, erythromycin, doxycycline and chloramphenicol

Question 8

cholera models

Answer 8

-humans are the only accurate model
-rabbit ligated ideal loops: Adult rabbit ileum is
divided into 5-10-cm segments by surgery, and live
bacteria or other test material injected into
individual loops. After 12-24h, animal is sacrificed;
loop length and fluid volume measured as ml/cm readout.
Infant mice (most common current model):
Newborn mice are inoculated with bacteria; at
sacrifice, stomach and entire gut are removed and
-weighed; gut wt/carcass wt = fluid accumulation
ratio. Bacteria can be cultured in vivo, and
competitive index determined.

Question 9

cholera toxin

Answer 9

-the toxin is the
major mediator of the cholera syndrome. Ingestion of only 5μg of purified toxin resulted in production of 1-6L of diarrheal stool in volunteers

Question 10

cholera pathogenicity

Answer 10

-All strains belonging to the species V. cholerae are not equally
pathogenic. Pathogenic strains are those strains capable of causing
disease.
* Toxigenic strains are those strains that possess the ability to
produce cholera toxin. Not all V. cholerae strains are capable of causing
cholera, because not all V. cholerae strains are capable of producing
cholera toxin.
* In order for a strain of V. cholerae to be pathogenic it must
encode the ctxA and ctxB genes, and it is dependent upon the
interaction between multiple genes including genes encoding one or
more virulence factors; these genes include the CTX phage and the TCP
pathogenicity island.
-essentially pathogenetic strain is the same as toxigenic strains

Question 11

virulence factors for cholera

Answer 11

Key Virulence Factors:

Cholera toxin (CT): Enterotoxin responsible for the symptoms of cholera.
Toxin-coregulated pili (TCP): Surface structure required for intestinal colonization.
Mobile Genetic Elements:

Both CTXΦ and TCP are mobile genetic elements.
CTXΦ (a filamentous bacteriophage) encodes:
ctxA and ctxB genes, which produce cholera toxin.
Toxigenic strains contain CTXΦ; non-toxigenic strains lack it.
Pathogenic Evolution:

Non-toxigenic strains can become toxigenic through horizontal gene transfer.
TCP not only aids pathogenicity but also acts as the receptor for CTXΦ.
CTXΦ Phage Characteristics:

Replicates as a plasmid in toxigenic strains.
Self-transmissible, producing DNA particles encoding the CTXΦ element.
In summary, CT and TCP are essential, coordinately regulated elements for V. cholerae virulence, with CTXΦ enabling toxigenicity through gene transfer and TCP supporting colonization and phage

Question 12

relationship between CTX0 and TCP

Answer 12

-in order for the transmission of CTXΦ to occur, the TCP must be present in the genome as it acts as the receptor for the CTXΦ.
*If the TCP is not expressed in a strain the CTXΦ will be unable to be transferred as the receptor required for transfer is not present.
*If the TCP is present in a non-toxigenic strain and the CTXΦ can be transferred by horizontal gene transfer. This can allow for the ctxA and ctxB
genes to be passed from toxigenic to non-toxigenic strains, which poses a
great threat as it can lead to the development of new toxigenic strains.
*This is an example of co-evolution of certain genetic elements that can lead
to the transduction of virulence genes.

Question 13

ctx genetic summary

Answer 13

-CTX genetic element
– Cholera toxin genes are
encoded on a lysogenic
filamentous bacteriophage
– The phage can infect strains
of V. cholerae missing toxin
genes
– The receptor for the phage is
Toxin co-regulated pilus (TCP)
– Transfer can occur in the
gastrointestinal tract or in the
aquatic environment

Question 14

cholera toxin mechanism of action

Answer 14

-Binding and Endocytosis: Cholera toxin (CTX) binds to intestinal cell surface receptors via its B subunits, triggering endocytosis of the toxin.

Activation of A1 Subunit: The A subunit of CTX is cleaved into A1 and A2 domains. The A1 domain becomes enzymatically active.

ADP-Ribosylation of G Protein: The A1 fragment enters the cytosol and ADP-ribosylates the Gsα subunit of the G protein, locking it in its active, GTP-bound form.

Stimulation of Adenylate Cyclase: The locked G protein continuously activates adenylate cyclase, leading to elevated cAMP levels.

Activation of CFTR: High cAMP levels activate the CFTR channel, causing a massive efflux of ions (e.g., chloride) and water from intestinal cells.

Result: This ion and water loss causes severe diarrhea, a hallmark of cholera infection.

Question 15

cholera:TCP

Answer 15

– Long filamentous pili termed the toxin-coregulated pilus (TCP) that
form bundles on bacterial surface is essential for colonisation
– Mutants lacking TCP are avirulent in human volunteers and in animal
models
– Genes necessary for production of TCP are organized in an operon that
contains 15 genes which is part of
Vibrio pathogenicity island (VPI)
* Major pilin subunit
* Assembly
* Secretion

Question 16

virulence factors regulation

Answer 16

-Transcription of the ctxAB operon is regulated by a number of environmental signals,
including temperature, pH, osmolarity, and certain amino acids.
-The ToxR protein is a regulatory protein which functions as an inducer in a
system of positive control.
ToxR interacts with ToxS in order to sense some change in the environment
and in response to induce the transcription of genes for attachment (pili
formation) and toxin production.
It is reasonable to expect that the environmental conditions that exist in the
GI tract (i.e., 37o temperature, low pH, high osmolarity, etc.), as opposed to
conditions in the extraintestinal (aquatic) environment of the vibrios, are
those that are necessary to induce formation of the virulence factors
necessary to infect.

Question 17

structure of cholera toxin

Answer 17

• key factor responsible for profuse diarroea
  -* It is composed of two polypeptides, A subunit and 5 B Subunits.
• The biological activity of CT is dependent on binding of the B pentamer to specific
  receptors on the eukaryotic cell.
• The B oligomer binds with high affinity exclusively to GM1 Ganglioside (Ganglioside
  is a molecule composed of glycosphingolipid with one or more sialic acids; it is a
  component of a cell plasma membrane).

Question 18

life cycle of pathogenetic vibrio cholerae

Answer 18

Toxigenic strains of Vibrio cholerae persist in aquatic environments alongside non-
toxigenic strains, aided by biofilm formation on biological surfaces and use of chitin as a
carbon and nitrogen source. On ingestion of these aquatic-environment-adapted
bacteria in contaminated food or water, toxigenic strains colonize the small intestine,
multiply, secrete cholera toxin and are shed back into the environment by the host in
secretory diarrhoea. The stool-shed pathogens are in a transient hyperinfectious state
that serves to amplify the outbreak through transmission to subsequent hosts.

Question 19

cholera vaccine development

Answer 19

-different vaccines have been tested and all failed
-Two recently developed oral vaccines for cholera are licensed and available in some
countries:
Dukoral®, Biotec AB (contains a mixture of inactivated V. cholerae bacteria and a
non-toxic component of the toxin), and Mutacol®, Berna (live attenuated).
Both vaccines appear to provide somewhat better immunity and fewer side-effects
than the previously available vaccine. However, neither of these two vaccines is
currently recommended for travellers

Question 20

salmonenella

Answer 20

-Enterobacteriaceae family
- Motile Gram-negative
facultative anaerobes
- Non-lactose fermenting
- Resistant to bile salts

Question 21

classification of salmonella

Answer 21

DNA homology shows only two species Salmonella enterica
(six subspecies) and S. bongori
· Most pathogens in S. enterica ssp. enterica. nomenclature still evolving
-Salmonella enterica subsp. enterica serotype Typhimurium
(Salmonella Typhimurium)
Salmonella serotypes are normally divided into two groups on the basis of host range: host adapted and ubiquitous

Question 22

salmonellosis

Answer 22

-generic term for disease.

Question 23

salmonella Eneritis

Answer 23

Most common form of salmonellosis with major
foodborne outbreaks and sporadic disease
 High infectious dose (108 CFU)
 Poultry, eggs, etc. are major sources of infection
 6-48h incubation period
 Nausea, vomiting, non-bloody diarrhoea, fever,
cramps, and headache common
 S. enterica serotypes (e.g., S. typhimurium)
 Zoonotic disease

Question 24

salmonella enteric fevers

Answer 24

S. typhi causes typhoid fever
S. paratyphi A, B and C cause milder form of
enteric fever
 Infectious dose ~ 106 CFU
 Fecal-oral route of transmission
· Person-to-person spread by chronic carrier
· Faecally-contaminated food or water
 10-14 day incubation period
 Initially signs of sepsis/bacteremia with sustained
fever (delirium) for > one week before abdominal
pain and gastrointestinal symptoms

Question 25

salmonella spread in the body

Answer 25

-survives in the low Ph of the stomach and evades the multiple defence of small intestine in order to gain access to the epithelium.
-salmonellae can
enter enterocytes and M cells, which transport them to the lymphoid cells (T and B)
-Once across the epithelium, Salmonella serotypes that are
associated with systemic illness enter intestinal macrophages and disseminate throughout
the reticuloendothelial system. By contrast, non-typhoidal Salmonella strains induce an early
local inflammatory response, which results in the infiltration of PMNs (polymorphonuclear
leukocytes) into the intestinal lumen and diarrhoea.

Question 26

salmonella in the gut

Answer 26

almonella infects gut epithelial cells, causing them to release chemoattractants like IL-8 and LTB4.
These chemoattractants attract neutrophils to the infection site in the gut lumen.
Neutrophils begin infiltrating within 1 hour and accumulate heavily by 5 hours.
This response helps fight the infection but can also cause inflammation and gut damage.

Question 27

salmonella virulenec factors

Answer 27

-Type III secretion systems and their effector molecules
2 different systems are present:
T3SS-1 is involved in promoting entry into intestinal
epithelial cells and induction of enteropathogenesis
T3SS-2 is involved in the ability of Salmonella to
survive inside macrophages
Flagella – help bacteria to move through intestinal
mucous
Capsule (for S. typhi and some strains of S. paratyphi)
Adhesins – both fimbrial and non-fimbrial Iron capturing ability

Question 28

Type III secretion systems in animal pathogens

Answer 28

-The type III secretion injectisome is a complex nanomachine
that allows bacteria to deliver protein effectors across eukaryotic cellular membranes.
Encoded on plasmids or “pathogenicity islands” and acts as a virulence factor.
Proteins Injected:
Translocators: Help deliver proteins into host cells.
Effectors: Alter host pathways to aid bacterial survival and infection.

Question 29

Type 3 needle complex

Answer 29

Structure:

The needle complex is a hollow, needle-like structure that spans across the bacterial membrane. It forms a molecular syringe that punctures the host cell membrane.
Function:

The needle serves as a conduit to inject bacterial effector proteins (which alter host cell processes) and translocator proteins (which create pores in the host cell membrane, allowing the effectors to enter).
Injection Process:

The bacteria use this needle to deliver specific effector proteins directly into the host cell’s cytoplasm or membrane. This is how bacteria like Salmonella can manipulate host cell functions to facilitate infection and evade the immune response.