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2.2 Microbiology > Denise - Bacillus > Flashcards

Denise - Bacillus Flashcards

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1
Q

Are bacillus endospore formers or not

A

They are endospore formers

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2
Q

What type of bacteria are bacilli

A

Gram positive bacilli
Aerobic
Endospore formers

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3
Q

What are the three bacillus species of interest

A

Bacillus cereus
Bacillus thurgenesis
Bacillus anthracis

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4
Q

How many Bacillus species are there

A

over 200 species identified

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5
Q

What infections does B. cereus cause

A

Extensively found in food and food processing environments

Opportunistic pathogen in other sites

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6
Q

Where is B. thuringenesis found

A

Insecticide

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7
Q

Where is B. anthracis found

A

Its a virulent pathogen of mammals

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8
Q

Where are all three Bacillus species found

A

Isolated from soil species

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9
Q

What is the B. cereus group

A

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

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10
Q

How are the B cereus group species similar to each other
(5)

A

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

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11
Q

Write about the clinical significance of Bacillus
(4)

A

Most species have little pathogenic potential

Opportunistic pathogens

Two species are clinically important (B. anthracis, B. cereus)

Major problem with contamination in the Lab

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12
Q

Write about B. cereus
(7)

A

Gram positive
Facultative anaerobe
Endospore former
Distinct colonial morphology
Large rods
Spores do not swell the sporangium
Most strains are motile and B-haemolytic

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13
Q

Write about the endospore forming aspect of B. cereus

A

Form endospores within a few days on commonly used agar media

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14
Q

What is the distinct colonial morphology of B. cereus

A

Ground glass appearance
Cells are large rods

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15
Q

Write about B. cereus reservoirs
(5)

A

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

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16
Q

Write about soil to food transmission of B. cerueus

A

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

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17
Q

What food is B. cereus found in
(5)

A

Rice
Dairy products
Spices
Dried foods
Vegetables

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18
Q

Write about B.cereus endospores
(7)

A

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

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19
Q

Write about B. cereus epidemiology
(5)

A

Notifiable to HPSC
About 1 case a year
Under estimated
No outbreaks in Ireland
Outbreaks reported in neonatal units in UK and France

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20
Q

Write about B. cereus foodborne disease
(5)

A

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

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21
Q

Write about B. cereus food association
(6)

A

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

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22
Q

What is the B. cereus diarrhoeal disease associated with

A

Proteinaceous foods:
- meat
- stews
- sauces
- milk
- vegetables
- fish

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23
Q

What foods are associated with the B. cereus emetic syndrome

A

Starchy foods:
- rice products
- potato
- pasta
- noodles
- pastry products

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24
Q

Write about the B. cereus diarrhoeal syndrome
(7)

A

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

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25
Q

What is the B. cereus diarrhoeal syndrome toxin

A

Diarrhoeagenic necrotising enterotoxin

Its a heat-labile enterotoxin

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26
Q

What is the incubation time for B. cereus diarrhoeal syndrome

A

6 hours

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27
Q

How long does B. cereus diarrhoeal syndrome last

A

12-24 hours

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28
Q

What are the three toxins associated with B, cereus diarrhoea

A

HBL (Haemolysin)
NHE (Non Haemolytic Entertoxin)
CytK

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29
Q

Write about HBL (Haemolysin)
(5)

A

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)

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30
Q

Write about the genetics behind Tripartite-3 component toxins (L2, L1 and B)

A

Encoded by the genes hblC, hblD and hblA

Cotranscribed from one operon

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31
Q

What does HBL (Haemolysin) actually do

A

Its dermonecrotic
Vascular permeability
Fluid accumulation in rabbit ileal loops
Cytotoxic and haemolytic

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32
Q

What percentage of B. cereus strains produce HBL (Haemolysin)

A

55%

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33
Q

What does HBL (Hameolysin) resemble

A

Forming toxin cytolysin A (ClyA)

34
Q

What is NHE

A

Non Haemolytic Enterotoxin

35
Q

Write about Non Haemolytic Entertoxin
(3)

A

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

36
Q

What percentage of B. cereus strains express Non Haemolytic Enterotoxin?

A

90% of all B. cereus strains

37
Q

What does Non Haemolytic Entertoxin cause

A

Rapid disruption of the plasma membrane
Formation of pores in the lipid bilayers

38
Q

What are the two B. cereus Beta barrel pore-forming toxins

A

CytK and Hlyll

39
Q

Write about the B. cereus Beta Barrel pore-forming toxins, CytK and Hlyll
(4)

A

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

40
Q

What does CytK do?

A

Dermonecrotic
Cytotoxic
Haemolytic

41
Q

What is emetic syndrome

A

Vomiting

42
Q

Write about emetic syndrome
(3)

A

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

43
Q

What toxin is responsible for emetic syndrome?

A

Emetic toxin “cerulide”

44
Q

How long does it take for onset of symptoms for emetic syndrome

A

0.5 - 6 hours after consumption of food

45
Q

Write about emetic toxin (cerulide)
(6)

A

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

46
Q

What is emetic toxin resistant to?
(3)

A

Autoclaving
pH
Protease

47
Q

Write about B. cereus oportunistic infections
(6)

A

Local infection

Bacteraemia and septicaemia

Central nervous system infections

Respiratory infections

Endocarditis (in drug abusers) and pericarditis

Immunocompromised patients Neonates

48
Q

What local infections does opportunistic B. cereus cause
(2)

A

Infections particularly of burns, traumatic or post-surgical wounds and of eye

Sever keratitis, endophthalmitis and panopthalmitis

49
Q

What central nervous system infections does B. cereus cause

A

Meningitis
Abscesses
Shunt-associated infections

50
Q

When might endocarditis by B. cereus be seen

A

In drug abusers

51
Q

Write about B. anthrax epidemiology
(6)

A

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

52
Q

What is the mortality rate of B. anthrax

A

<1-20%

But there is only 1-2 cases per year USA

53
Q

Write about anthrax infections
(5)

A

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

54
Q

Describe how anthrax infections develop
(4)

A

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

55
Q

Write about anthrax infection severity
(4)

A

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

56
Q

Write about B. anthrax cutaneous anthrax
(6)

A

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

57
Q

Write about intestinal anthrax

A

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

58
Q

Write about pulmonary anthrax

A

Endospores are carried from the lungs by macrophages to the lymphatic system where they germinate and multiply to produce a fatal septicaemia

59
Q

Write about Bacillus bioterrorism

A

2011
Sent through US Postal Service

60
Q

Write about B. anthracis - Virulence: Capsule
(6)

A

B. anthracis produces a number of virulence factors

The poly D glutamic acid capsule
- antiphagocytic
- essential for virulence

Plasmid
pXO2 caosule

61
Q

What are the two B. anthrax toxins associated with virulence

A

Oedema factor -> increases CAMP
Lethal factor -> metalloprotease tissue necrosis

62
Q

Write about the 2 plasmid encoded exotoxins, oedema factor and lethal factor

A

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)

63
Q

What are some safety considerations for laboratory investigation behind Bacillus species

A

B. anthracis is a Class 3 pathogen

Category 3 Lab facilities are required

64
Q

What clinical samples is B. anthracis seen in?
(3)

A

Cutaneous (95%) -> tissue

Gastrointestinal (5%) -> faeces

Inhalation (<1%) - Lung aspirates, pleural fluid, tissue samples, blood, CSF

65
Q

How is B. anthraxis inoculated

A

Blood agar
PLET agar
Incubates at 37 degrees for 24-48 hours

66
Q

What is PLET agar

A

Polymuxin-lysozyme-EDTA- thallous acetate agar

67
Q

What samples might be seen for B. cereus

A

Numerous samples
Environmental samples
Food

68
Q

What media is B. cereus isolated on

A

Isolation media
Blood agar
Selective agar
- Bacillus cereus selective agar (PEMBA)

69
Q

Write about B. cereus on PEMBA agar

A

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

70
Q

What is the basic characterisation for Bacillus

A

Gram positive baciili (large box car ends Central spores)
KoH negative
Catalase positive
Oxidase negative

71
Q

What confirmatory tests are put up on day 2 and 3 for Bacillus species

A

Biochemical identification
Maldi: Not Disc enough
Toxin detection

72
Q

Write about the detection of B. cereus emetic toxin
(5)

A

Cell culture assays using Hep-2 cells

These lack specificity

HPLC-MS analysis can be done

Maldi Tof MS

Real time PCR ces genes

73
Q

What is meant by cell culture assays using Hep-2 cells to detect B. cereus emetic toxin

A

Using Hep 2 cell lines with culture filtrates
Cause vacuolization of this cell line
Toxin induced cell damage

74
Q

Why is HPLC-MS not really used for emetic toxin detection

A

Laborious and costly

75
Q

Write about detection of B. cereus cytotoxin NHE

A

TECRA-BDE kit is used

76
Q

What is TECRA -BDE kit

A

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

77
Q

Write about detection of B. cereus cytotoxin HBL detection

A

The Oxoid BCET-RPLA kit is used

78
Q

What is the Oxoid BCET-RPLA kit

A

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

79
Q

What are the Duopath Cereus Enterotoxins: HBL and NHE

A

Immunochromatographic rapid test based on gold labelled antibodies

80
Q

How are B. anthracis toxins detected

A

PCR
ELISA to detect in serum or pleural fluid

81
Q

Write about the treatment and prevention of anthrax

A

Vaccination for workers at high risk of exposure
Ciprofloxacin for cutaneous
Ciprofloxacin and rifampicin and vancomycin for inhalation

2.2 Microbiology (60 decks)

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