Gram negative coccobacilli, Brucella, Francisella, Pasteurella, Yersinia, Bordatella

Question 1

What are the genuses of Gram negative Coccobacilli

Answer 1

Haemophilus and Bordetella

H. influenzae

H. parainfluenzae

H. aegyptius

H. ducreyi

B. pertussis

B. parapertussis

Question 2

Haemophilus influenzae

Morphology

Culture

Answer 2

A Gram negative cocco-bacillus (long), 1.5uM coccobacilli in pairs mostly or short chains.

is a fastidious bacteria: this blood loving (heamophil) bacteria requires factor X, Heme and factor V, NAD in its medium. So it must be grown on chopped-meat enriched chocolate agar.

Or it can be grown as a satellite colony around a Staphylococcus aureus streak

facultative anaerobe.

Catalase: negative

Oxidase: negative

Urease: positive

Colonies: tiny clear colonies in blood S. a satellite culture, small gray colonies on chocolate agar.

UsingSims-discs on simple agar. They grow near factors X+V, but not X or V alone.

Question 3

Haemophilus influenzae, Virulence factors

Answer 3

Capsule, serotypes a-f -

capsule b = bad H.i b is the most common invasive type which causes severe disease in children.

There is a childhood vaccine against the type b

There are also many species of uncapsulated H. influenzae and these must be identified by geneotype. Unencapsulated forms are much less invasive and cause only local infections. Are often opportunistic infections in patients already suffering from other respiratroy disease.

IgA protease

Surface antigens: OMPs, outer membrane proteins and LPS

Hemolysins

Cytolethal distending toxin (CDT) -

Question 4

Heamophilus influenzae

Habitat

Transmission

Answer 4

An obligate pathogen

Reservior is the human respiratory system and nasopharynx

Transmitted via respiratory droplets

Question 5

Heamophilus influenzae

Clinical diseases

Diagnosis

Answer 5

Upper respiratory infections:

Nasopharyngitis, Sinusitis,

Epiglottitis - represents severe chocking danger,

Otitis media - middle ear infection with a danger of eardrum perforation.

Lower respiratory infections: Bronchitis, Pneumonia

Septic Arthritis - infection of a synovial joint, along with fever

Invasive severe diseases:

Cellulitis - especially periorbital cellulitis causing swelling of the eyelids in infants/children.

Meningitis

Vasculitis

Sepsis - secondary DIC, gangrene of extremities

Question 6

H. influenzae

Diagnosis

Treatment

Answer 6

Diagnosis: Samples from infection site, also from CSF

Diagnose by culture characteristics

by Antigen Latex-agglutination for capsule (a-f variants)

by direct immunofluorescent labeling of H. i b antigens in the CSF

rtPCR from blood and CSF samples

Treatment:

Ampicillin and 3rd gen Cephalosporins

or

Ampicillin and Aminoglycosides

but as usual do an antibiogram, 5-40% are penicillin resistant, and a beta-lactamase inhibitor should be used as well.

Active immunisation of children against HiB by vaccine

Activie immunisation of pregnant mothers to prevent infection during birth

Question 7

Haemophilus ducreyi

Morphology

Culture

Answer 7

Gram negative coccobacillus/bacillus (rod) in long chains.

demanding requires heme (factor X), but not NAD (V) (chocolate agar).

Small whitish colonies

Question 8

H. ducreyi

Clinical diseases

Treatment

Answer 8

Causes only the sexually transmitted disease chancroid.

Chancroid = Ulcus molle = ulcer soft (as opposed to ulcus durum, hard ulcer of syphilis)

Treatment:

Macrolide or

3rd generation Cephalosporin

Question 9

What are the 4 major species of Haemophilus bacteria?

What are the diseases they cause?

Answer 9

H. influenziae - many, from mild sinusitis to severe sepsis, meningitis

H. ducreyi - the STD Chancroid

H. aegyptius - Brasilian purpuric fever

H. parainfluenzae - Pharyngitis, Endocarditis, Conjunctivitis

Question 10

Bordetella pertussis

Morphology

Culture

Answer 10

Gram negative coccobacillus (short), 1uM in pairs

Special medium Bordet-Gengou medium - contains potato extract, glycerol, 20% sheep blood, and antibiotics to inhibit normal flora

• *Distinct mercury like appearance** on this special medium. Small round blue/silver/gray on the 20% blood agar BG-medium.
• *No hemolysis**

Or on Charcoal agar supplemented with defibrinated horse blood. Charcoal, along with starch, neutralizes fatty acids and peroxides, which are toxic to Bordetella.

On Charcoal agar, the pertussis colonies are solid white.

Question 11

Bordatella pertussis

Clinical diseases

Treatment

Answer 11

“Pertussis” = violent cough

Bordatella pertussis causes whooping cough.

Question 12

Bordetella pertussis

Virulence factors

Answer 12

Endotoxins

Capsule

Fimbriae

Filamentous hemagglutinin - mediates attachment of the bacteria to the respiratory epithelium.

Outer membrane proteins (OMP)

LPS

Pertactin - A major virulence factor: Adhesion to tracheal respiratory endothelial cells.

Exotoxins:

Pertussis Toxin - Has a pore forming region and another regionwhich activates G_s proteins, which then in turn activate cellular adenylate cyclase, to generate cAMP. Generates cilliary stasis, allowing for enhances cilia binding by filamentous hemagglutinin.

Adenylate-cylcase toxin - a toxin composed of a pore forming domain and its own adenylate cyclase domain. enters cells that B. p is bound to, and causes drastic increases in cAMP. Impairs chemotaxis, as well as inhibiting peroxide and superoxide synthesis, inhibiting immune system.

Tracheal cytotoxin - damages tracheal epithelial cells, damages their cilia, impeding their ability to move bacterial outward from resp. tract.

Dermatonecrotic toxin - activates cellular Rho-GTPase.

Question 13

Bordetella pertussis

Habitat

Transmission

Answer 13

Humans are only reservoir.

Very contagious and spread by respiratory route during the first two stages of the disease: Catarrhal stage and Paroxysmal stage

Question 14

Bordatella pertussis

Clinical disease

Answer 14

Disease: Whooping cough, incubation followed by 3 stages:

Incubation: 7-10 days

Catarrhal stage: 1-2 weeks of throat/nasal type infection and possibly conjunctivitis. Fever, malaise and loss of appetite.

Paroxysmal stage: 2-4 weeks. Fever subsides, nonproductive whooping cough develops. 15-25 attacks a day of a several minutes of coughing and wheezing breath. Hypoxia and cyanosis occurs. Neck veins and eyelids bulge, capillaries in the sclera may rupture. Vomitting often occurs after the attack.

Convalescent stage: 3-4 weeks or longer: Coughing attacks decrease. no longer contagious. Secondary complications often occur during this stage such as pneumonia or encephalopathy.

Question 15

Bordetella pertussis

Diagnosis

Answer 15

Diagnosis:

Cough directly onto the Bordet-Gengou or Charcoal supplemented blood agar.

Or swabbed with a calcium alginate swab, bc the pertussis will not grow on cotton.

Slide agglutination with antibodies against B. pretussis

Direct IF immunolabeling on direct plate smears

ELISA for circulating anti pertussis antibodies

Widal’s type tube agglutination for circulating anti-pertussis antibodies using laboratory pertussis strains.

rtPCR

Question 16

Bordatella pertussis

Treatment

Prevention

Answer 16

Treatment:

During the prodromal or cattharal stage, Erythromycin (macrolide) or Ampicillin can shorten disease or lessen symptoms.

Resistant to penicillin G.

Treatment during the paroxysmal stage (severe coughing stage) does not alleviate any symptoms, but may decrease the infectivity and bacterial shedding of the patient.

Primarily supportive

Prevention:

Active immunization with neutralized B pertussis as part of the DPT vaccine (Diptheria Pertussis Tetanus)

Acellular vaccine: DaPT vaccine (Diptheria toxoid - “a” for acellular, pertussis, and tetanus.

Question 17

Differential diagnosis of

• Bordetella pertussis*
• B. parapertussis*
• and*
• B. bronchiseptica*

Answer 17

B pertussis: Requires special agar, Bordet-Gengeu. Others will grow on normal or special agar. Oxidase positive, and Urease negative

B parapertussis: Oxidase negative, Urease positive, and will grow on normal agar.

B bronchiseptica: Is motile and has cillia, the others are not. It is also positive for all the other tests.

Question 18

Brucellae genus

Morphology

Culture

Answer 18

Gram negative coccobaccili, short coccobacilli

Fastidious requires more than 24 hours and nutrient rich agar containing Serum and Glycine supplement.

Pinpoint smooth shiny colonies. Non-hemolytic at 48 hours

Facultative anaerobic and Facultative intracellular. Is cultured most effectively anaerobically.

*Though they are slow growing, Brucella are hearty, and are easy to grow and maintain in animal stocks. They are also easily disseminated through water droplets and highly infective. Because of all this they are categorized and tracked well as a potential bioterrorism agent.

Question 19

What are the Brucella species ?

What is their habitat?

How are they transmitted?

Answer 19

They are all zoonotically transmitted

by ingestion of infected animal tissue or milk, or direct skin through skin lesions or eye conjunctiva.

After injestion or skin contact, brucella penetrates through skin lesions or GI mucosa and enters the lymphatics. Where the bacteria proliferate by intracellular growth within bacrophages. This is followed by blood infection and widespread organ infiltration.

Reserviors are animals. Patients are usually animal related-workers.

• B. abortus* - causes abortions in cows
• B. melitensis* - infects goats
• B suis* - pigs
• B canis* - dogs

Every Brucella species causes the same type of disease, Brucellosis in humans

Question 20

Brucella

Clinical disease

Answer 20

Brucellosis

Fever and chills aka “undulant fever”, RES

loss of appetite, backache, headache, sometimes lymphadenomas.

Orchitis - testicular inflammation, swelling

Carditis

Arthritis and joint swelling often of knees, feet

Symptoms are long lasting from months to years

But fortunately disease is rarely fatal

Question 21

Brucella

Diagnosis

Treatment

Prevention

Answer 21

By clinical specimen sampling of:

Sputum, urine, blood-culture, CSF, bone marrow, and lymph node punctate:

Direct smear of intracellular gram negative coccobacilli

Culturing is slow, so serology methods should be used.

Serology for Brucella IgM, Tube agglutination

IgM chromatography

ELISA

Treatment:

Doxycyclin, Rifampicin, Streptomycin, Tetracycline

Prevention:

No specific prevention exists. Controlling animal health.

Question 22

Fancisella tularensis

Habitat

Transmission

Answer 22

Francis the rabit in the Tulip field.

It is harbored by over 100 species of animals and insects, and is zoonotically transmitted by handling carrier animals or being bitten by an insect vector, fly, tick.

It is extremely virulent will start an infectious lesion wherever it makes contact.

If it is on the skin, –> Ulceroglandular tularemia

If it is inhaled (inhaling aerosolized particles while skinning the animal, inhaling a fly) –> Pneumonic tularemia

If it hits the eye –> oculoglandular tularemia

Ingested –> typhodal tularemia

But, there is no human to human transmission.

Question 23

Francisella Tularensis

Clinical disease

Diagnosis

Answer 23

Tularemia is clinically very similar to the bubonic plague. with the one exception that skin ulcers usually do not develop in the plague.

Skin ulceration with well defined boundaries.

Lymphadenoma, granuloma

Since the bacteria is so infectious it is not cultured.

It is confirmed by measuring the titers of circulating antibodies against F. tularensis.

and rtPCR of clinical samples.

Question 24

Francisella tularensis

Treatment

Question 25

Francisella tularensis

Morphology

Culture

Answer 25

A gram negative bacillus/rod

Culturing it is Prohibited, because it is so infectious

When it is cultured on cysteine heart agar with blood, the cultures are opalescent (yellow, multicolored) and do not discolor the medium.

Question 26

Yersinia pestis

morphology

culture

Answer 26

The boubonic plague (boubon - groin- inguinal node swelling)

gram negative bacilli/rods with bipolar staining, vaculolated middle part

Question 27

Yersinia pestis

Virulence factors

Answer 27

F1 VW, Feul injected VW bug.

Capsule protein F1: inhibits phagocytes

V antigen (protein) and W antigen (lipoprotein) are unique Yersinia antigens.

V antigen, antiphagocytic, unknown mechanism.

W antigen, an endotoxin. Unknown mechanism

Exotoxins:

Plasminogen activator

Toxin - causes the lethality in mice

Question 28

Yersinia pestis

Habitat

Transmission and Clinical disease development

Answer 28

Its reservior is wild rodents, rats.

Transmitted to domestic animals and humans by fleas, anthropo-zoonosis

also by rat bites

In the flea, the bacteria’s coagulase blocks its esophagus, causing it to regurgitate bacteria into the wound it makes when it bites a human.

Proliferates within monocytes (is killed by PMN cells), and is carried to the lymph nodes, causing the swollen, red, painful lymphadenoma.

In the lymph nodes, causes necrosis and hemorrhageic inflammation, spreading to the blood stream. –> speticemia,

From the blood stream –> meningitis, pluero-pericarditis, hemorrhagic pneumonia, DIC, gangrene of extremities.

Pneumonia –> coughing, respiratory droplets containing blood and virus, human-human transmission.

Importantly, lymphadenoma does not always occur (in up to 25% of cases) so if someone has potential exposure it should be treated rapidly because it is fatal in 75% of untreated cases

Question 30

Yersinia pestis

Diagnosis

Treatment

Answer 30

Clinical samples, from the lymph nodes, sputum, blood cultures, CSF

Direct smear: Geimsa stain, immunofluorescent stain,

Wayson’s stain produces the bipolar rod stain “hairpin” appearance.

rt PCR

tube agglutination for circulating antibodies.

Culturing is usually Prohibited. Must be sent to specific approved labs for culturing, it is a regulated bioterror organism.

on blood agar, tiny, grey, viscous colonies.

lactose , urease , oxidase negative

ONPG positive

Treatment:

Doxycyclin, Streptomycin.