Denise - Bacillus Flashcards
Are bacillus endospore formers or not
They are endospore formers
What type of bacteria are bacilli
Gram positive bacilli
Aerobic
Endospore formers
What are the three bacillus species of interest
Bacillus cereus
Bacillus thurgenesis
Bacillus anthracis
How many Bacillus species are there
over 200 species identified
What infections does B. cereus cause
Extensively found in food and food processing environments
Opportunistic pathogen in other sites
Where is B. thuringenesis found
Insecticide
Where is B. anthracis found
Its a virulent pathogen of mammals
Where are all three Bacillus species found
Isolated from soil species
What is the B. cereus group
B. cereus sensu lato
Genetically highly homogenous
A subdivision of the genus Bacillus
Six recognised species
Closely related by genome sequence
Same phylogenetic cluster
Differentiated by phenotypic characteristics and pathological properties
Many distinguishing features between the species are encoded by genes located on plasmids
How are the B cereus group species similar to each other
(5)
B. anthracis, B. cereus, B. thuringiensis
Closely related by genome sequence
Same phylogenetic cluster
Differentiated by phenotypic characteristics and pathological properties
Many distinguishing features between the species are encoded by genes located on plasmids
Write about the clinical significance of Bacillus
(4)
Most species have little pathogenic potential
Opportunistic pathogens
Two species are clinically important (B. anthracis, B. cereus)
Major problem with contamination in the Lab
Write about B. cereus
(7)
Gram positive
Facultative anaerobe
Endospore former
Distinct colonial morphology
Large rods
Spores do not swell the sporangium
Most strains are motile and B-haemolytic
Write about the endospore forming aspect of B. cereus
Form endospores within a few days on commonly used agar media
What is the distinct colonial morphology of B. cereus
Ground glass appearance
Cells are large rods
Write about B. cereus reservoirs
(5)
Ubiquitous
Soils, sediments, dust and plants
Spores can be passively spread and can be found outside natural habitats
Germinates, grows and sporulates in soil, thus demonstrating a saprophytic life cycle
Has been reported to be present in stools of healthy humans at varying levels
Write about soil to food transmission of B. cerueus
Isolated from a range of different foods
Cross-contamination can distribute spores or cells to other foods, such as meat products
At harvest cells or spores may accompany plant material into food production areas and establish on food-processing equipment
What food is B. cereus found in
(5)
Rice
Dairy products
Spices
Dried foods
Vegetables
Write about B.cereus endospores
(7)
Heat treatment
Food processing
Sanitisers
Spore can adhere to surfaces in the food processing lines
Not removed by regular cleaning of surfaces
Role in biofilm persistence in food industry equipment such as dairy pipelines
The biofilm protects spores and vegetative cells against inactivation by sanitisers
Write about B. cereus epidemiology
(5)
Notifiable to HPSC
About 1 case a year
Under estimated
No outbreaks in Ireland
Outbreaks reported in neonatal units in UK and France
Write about B. cereus foodborne disease
(5)
Two distinct foodborne disease types, emetic and diarrhoeal
Mild and self-limiting
B. cereus was established as an organism of foodborne disease in the 1950s
Outbreaks of the diarrhoeal type of disease in hospitals in Norway in 1947
The emetic syndrome was first identified after several outbreaks caused by eating cooked rice in the UK in early 1970s
Write about B. cereus food association
(6)
Wide environmental distribution of spores allows B. cereus access to a variety of foods
Proteinaceous foods associated with the diarrhoeal disease
The emetic syndrome is associated with starchy foods
No legislation requires screening of food items for this pathogen contamination
Dried infant formulae have an established maximum limit of 50 CFU/g
Outbreaks on neonatal units
What is the B. cereus diarrhoeal disease associated with
Proteinaceous foods:
- meat
- stews
- sauces
- milk
- vegetables
- fish
What foods are associated with the B. cereus emetic syndrome
Starchy foods:
- rice products
- potato
- pasta
- noodles
- pastry products
Write about the B. cereus diarrhoeal syndrome
(7)
Toxicoinfection
Vegetative cells, ingested as viable cells or spores producing protein enterotoxins in the small intestine
Heat-labile enterotoxin
Diarrhoeagenic necrotising enterotoxin
Onset of watery diarrhoea and cramps within 6-15 hours post consumption of contaminated food
Incubation time is over 6 hours with a normal range between 8-16 hours
Duration of symptoms is 12-24 hours
What is the B. cereus diarrhoeal syndrome toxin
Diarrhoeagenic necrotising enterotoxin
Its a heat-labile enterotoxin
What is the incubation time for B. cereus diarrhoeal syndrome
6 hours
How long does B. cereus diarrhoeal syndrome last
12-24 hours
What are the three toxins associated with B, cereus diarrhoea
HBL (Haemolysin)
NHE (Non Haemolytic Entertoxin)
CytK
Write about HBL (Haemolysin)
(5)
Tripartite-3 component toxin (L2, L1 and B)
B is for binding, L1 and L2 are lytic components
Cytotoxic and haemolytic
55% of all B. cereus strains
Tertiary structure resemblance with the pore-forming toxin cytolysin A (ClyA)
Write about the genetics behind Tripartite-3 component toxins (L2, L1 and B)
Encoded by the genes hblC, hblD and hblA
Cotranscribed from one operon
What does HBL (Haemolysin) actually do
Its dermonecrotic
Vascular permeability
Fluid accumulation in rabbit ileal loops
Cytotoxic and haemolytic
What percentage of B. cereus strains produce HBL (Haemolysin)
55%
What does HBL (Hameolysin) resemble
Forming toxin cytolysin A (ClyA)
What is NHE
Non Haemolytic Enterotoxin
Write about Non Haemolytic Entertoxin
(3)
Large food poisoning outbreak in Norway in 1995 caused by the hbl-negative B. cereus strain
Produced by 90% of all B. cereus strains
NHE is a three-component toxin with cytotoxic activity towards epithelial cells
What percentage of B. cereus strains express Non Haemolytic Enterotoxin?
90% of all B. cereus strains
What does Non Haemolytic Entertoxin cause
Rapid disruption of the plasma membrane
Formation of pores in the lipid bilayers
What are the two B. cereus Beta barrel pore-forming toxins
CytK and Hlyll
Write about the B. cereus Beta Barrel pore-forming toxins, CytK and Hlyll
(4)
Both single component protein toxins
This toxin family includes B-toxin of C. perfringens and alpha-haemolysin of S. aureus
Transmembrane pore at target cell surface
CytK is a single 34 kDa protein with dermonecrotic, cytotoxic and haemolytic activities
What does CytK do?
Dermonecrotic
Cytotoxic
Haemolytic
What is emetic syndrome
Vomiting
Write about emetic syndrome
(3)
Outbreaks after eating cooked rice in the UK in the 1970s
Intoxication
- ingestion of a heat stable toxin (emetic toxin “cerulide”) produced in food before ingestion
Nausea and vomiting within 0.5-6 hours after consumption of food: Rapid onset of symptoms
What toxin is responsible for emetic syndrome?
Emetic toxin “cerulide”
How long does it take for onset of symptoms for emetic syndrome
0.5 - 6 hours after consumption of food
Write about emetic toxin (cerulide)
(6)
Ring structure of 3 repeats of 4 amino acids
Produced by a nonribosomal peptide synthetase
Encoded by the 24-kb cereulide synthetase (ces) gene cluster
Plasmid
Mechanism unkown -> receptor mediated
Resistant to autoclaving, pH and protease
What is emetic toxin resistant to?
(3)
Autoclaving
pH
Protease
Write about B. cereus oportunistic infections
(6)
Local infection
Bacteraemia and septicaemia
Central nervous system infections
Respiratory infections
Endocarditis (in drug abusers) and pericarditis
Immunocompromised patients Neonates
What local infections does opportunistic B. cereus cause
(2)
Infections particularly of burns, traumatic or post-surgical wounds and of eye
Sever keratitis, endophthalmitis and panopthalmitis
What central nervous system infections does B. cereus cause
Meningitis
Abscesses
Shunt-associated infections
When might endocarditis by B. cereus be seen
In drug abusers
Write about B. anthrax epidemiology
(6)
Zoonotic disease
Exposure to animals (sheep, goats, horses)
Exposure to animal products
Difficult to eradicate from endemic areas due to endospores
Cutaneous anthrax account for 95-99% of human cases worldwide
<1-20% mortality rate; 1-2 cases per year USA
What is the mortality rate of B. anthrax
<1-20%
But there is only 1-2 cases per year USA
Write about anthrax infections
(5)
Incubation period of 2-5 days
Small pimple or papule develops over 24 hours
The lesion is surrounded by oedema
May be necrosis
Not purulent
Describe how anthrax infections develop
(4)
Small pimple or papule develops
A ring of vesicles develops around the papule
Ulcerates, dries and blackens into a characteristic eschar
This enlarges and becomes thick and adherent to underlying tissue over the ensuing weeks
Write about anthrax infection severity
(4)
Secondary sequelae to cutaneous anthrax include cellulitis and meningitis
About 20% of untreated cases are fatal
The mortality rate drops to zero if patients are treated
Write about B. anthrax cutaneous anthrax
(6)
Endospores enter through a cut, they germinate and vegetative cells multiple
Acute host inflammatory response - capsule prevents phagocytosis
Exotoxin release
Invasive damage
Rapid development of a malignant pustule
Associated with cases where humans handle infected animals and/or animal products
Write about intestinal anthrax
Cutaneous anthrax on intestinal mucosa
It is sometimes accompanied by gastroenteritis
Low level germination at one site leads to massive effusion, mucosal oedema and necrotic lessions
Write about pulmonary anthrax
Endospores are carried from the lungs by macrophages to the lymphatic system where they germinate and multiply to produce a fatal septicaemia
Write about Bacillus bioterrorism
2011
Sent through US Postal Service
Write about B. anthracis - Virulence: Capsule
(6)
B. anthracis produces a number of virulence factors
The poly D glutamic acid capsule
- antiphagocytic
- essential for virulence
Plasmid
pXO2 caosule
What are the two B. anthrax toxins associated with virulence
Oedema factor -> increases CAMP
Lethal factor -> metalloprotease tissue necrosis
Write about the 2 plasmid encoded exotoxins, oedema factor and lethal factor
Both are AB toxins (activity Binding)
The binding unit is shared by both toxins and is called a protective antigen
Mediated cell entry of both toxins
pXO1 (anthrax toxin)
What are some safety considerations for laboratory investigation behind Bacillus species
B. anthracis is a Class 3 pathogen
Category 3 Lab facilities are required
What clinical samples is B. anthracis seen in?
(3)
Cutaneous (95%) -> tissue
Gastrointestinal (5%) -> faeces
Inhalation (<1%) - Lung aspirates, pleural fluid, tissue samples, blood, CSF
How is B. anthraxis inoculated
Blood agar
PLET agar
Incubates at 37 degrees for 24-48 hours
What is PLET agar
Polymuxin-lysozyme-EDTA- thallous acetate agar
What samples might be seen for B. cereus
Numerous samples
Environmental samples
Food
What media is B. cereus isolated on
Isolation media
Blood agar
Selective agar
- Bacillus cereus selective agar (PEMBA)
Write about B. cereus on PEMBA agar
PEMBA is selective and differential
B. cereus does not ferment mannitol
B. cereus produces lecithinase
B. cereus is resistant to polymixin
Colonies on PEMBA agar have peacock blue colonies
Cannot differentiate between B. cereus and thuringenesis
What is the basic characterisation for Bacillus
Gram positive baciili (large box car ends Central spores)
KoH negative
Catalase positive
Oxidase negative
What confirmatory tests are put up on day 2 and 3 for Bacillus species
Biochemical identification
Maldi: Not Disc enough
Toxin detection
Write about the detection of B. cereus emetic toxin
(5)
Cell culture assays using Hep-2 cells
These lack specificity
HPLC-MS analysis can be done
Maldi Tof MS
Real time PCR ces genes
What is meant by cell culture assays using Hep-2 cells to detect B. cereus emetic toxin
Using Hep 2 cell lines with culture filtrates
Cause vacuolization of this cell line
Toxin induced cell damage
Why is HPLC-MS not really used for emetic toxin detection
Laborious and costly
Write about detection of B. cereus cytotoxin NHE
TECRA-BDE kit is used
What is TECRA -BDE kit
TECRA bacillus diarrhoeal enterotoxin visual immunoassay
Detects the NheA component of the Nhe toxin by an ELISA sandwich testT
The sensitivity reported by the manufacturer is >1 ng mL prepared sample
The kit is intended for use on foods and environmental samples
Write about detection of B. cereus cytotoxin HBL detection
The Oxoid BCET-RPLA kit is used
What is the Oxoid BCET-RPLA kit
Semi quantitative
Detects by reversed antibody agglutination
The L2 component of Hbl in foods and in culutres of B. cereus
The kit is intended for use on foods and environmental samples
What are the Duopath Cereus Enterotoxins: HBL and NHE
Immunochromatographic rapid test based on gold labelled antibodies
How are B. anthracis toxins detected
PCR
ELISA to detect in serum or pleural fluid
Write about the treatment and prevention of anthrax
Vaccination for workers at high risk of exposure
Ciprofloxacin for cutaneous
Ciprofloxacin and rifampicin and vancomycin for inhalation
