Brucella, Campylobacter, Helicobacter (Ch2)

Question 1

What are the characteristics of Brucella spp.?

Answer 1

• Gram negative
• Aerobic
• Slow growing
• Coccobacilli
• Lack capsule, Flagella, Endospores, Native plasmids
• Zoonotic

Question 2

How is brucellosis transmitted?

Answer 2

Transmission can occur through:

1. Inhalation of contaminated aerosols
2. Ingestion of unpasteurized dairy products
3. Direct contact with infected animals or their secretions through cuts or
   abrasions in the skin or conjunctival sac.
4. Contact with fluids from infected animals (sheep, cattle, goats, pigs, or
   other animals)

Question 3

Name all Brucella species and their associated animal hosts

Answer 3

• Brucella melitensis: Associated with goats and sheep
• Brucella abortus: Associated with cattle
• Brucella suis: Associated with swine and caribou
• Brucella canis: Associated with dogs and foxes
• Brucella inopinata
• Brucella ceti: Associated with marine mammals, specifically cetaceans (whales, dolphins, and porpoises)
• Brucella pinnipedialis: Associated with marine mammals, specifically pinnipeds (seals and sea lions)

Question 4

What is the characteristic manifestation of Brucellosis disease

Answer 4

• Undulant fever (38-41°C)
• Malta fever
• Mediterranean fever
• Gibraltar fever

Question 5

What is the pathogenesis of brucellosis?

Answer 5

Macrophage phagocytes brucella bacterium → Bactetia inhibits phagosome-lysosome fusion → Bacteria survives and multiples inside macrophages → Bacteria is carried to the spleen, bone marrow, lymph nodes, and kidneys → Bacteria multiples in the cells of the reticuloendothelial system → Formation of small granulomas (macrophage aggregates) and the release of brucella bacteria in circulation → Septicemia

Question 6

How does Brucella’s LPS compare to other gram-negative bacteria’s LPS?

Answer 6

Brucella LPS (Non classical LPS) is considered less toxic compared to LPS from other Gram-negative bacteria (Classical LPS). It exhibits lower endotoxic activity, lower pyrogenicity (fever-inducing) and is a weak inducer of interferons (IFs) and tumor necrosis factor (TNFs)

Question 7

What are the main reasons Brucella causes undulant fever?

Answer 7

1. Macrophage replication is slow
2. Brucella LPS is less toxic (delayed immune response)

Question 8

What are the clinical symptoms of brucellosis?

Answer 8

1) Gastrointestinal tract symptoms (70%)
> Diarrhea, abdominal pain, nausea, etc.

2) Osteomyelitis
> Large joints (knees & joints) are commonly affected, especially in children

3) Respiratory tract symptoms (uncommon)
> Range from flu-like symptoms with normal X-rays to bronchitis, pneumonia, lung nodules, lung abscesses

4) Genitourinary disease (2-40% of males)
> *Especially epididymo-orchitis (inflammation of the testicles)

5) Neurobrucellosis (1-2%)
> usually meningitis

6) Endocarditis (1%)
> *2/3 cases occur on previously damaged heart valves, usually left-sided endocarditis *

7) Hepatic abscesses (1%)

8) Abortion

9) Other less common complications include pneumonitis, pleural effusion or abscess involving the spleen, thyroid, or epidural space

Question 9

What specimen is collected for the diagnosis of brucellosis?

Answer 9

Blood, BM, urine, CSF, synovial fluid (joints), liver biopsy, lymph nodes biopsy

Question 10

Explain the diagnostic steps taken to identify Brucella

Answer 10

1. Culture in enriched blood agar for long incubation period of at least three wells (slow growing bacteria)
2. Identification
   > Oxidase test (positive)
   > Urease test (positive)
   > Agglutination assay (agglutination =presence of anti-Brucella antibodies) (determines species of Brucella)
   > Serology
   > PCR

Question 11

Which tests are relied on the most for the diagnosis of Brucella?

Answer 11

Serology (Wright agglutination test) and PCR

Question 12

What is the basis of Wright agglutination test

Answer 12

-ve to a titer of 1:40 or less → normal healthy population
A titer of 1:80 or more → clinically significant

Question 13

What is required in a serological test to diagnose acute infection?

Answer 13

A 4-fold or greater increase in titer between acute and convalescent phase is indicative of acute infection.

Question 14

What required in serological tests to supports the diagnosis of brucellosis?

Answer 14

It is generally agreed that a titer of >1:160 in the presence of a compatible illness supports the diagnosis of brucellosis.

Demonstration of a fourfold or greater increase in agglutinating antibodies over 4
to 12 weeks provides even stronger evidence for the diagnosis.

Question 15

What are the prevention measures taken for brucellosis?

Answer 15

• Systemic identification and elimination of infected
herds and animal vaccination
• Avoid unpasteurized dairy products
• Good laboratory practice and safety
• Live attenuated B. abortus and B. melitensis vaccines:
used for animals (Absence of human vaccine)

Question 16

What are the characteristics of Campylobacter species?

Answer 16

• Gram-negative comma/S-shaped rods
• Motile with single polar flagellum
• Non-spore forming
• Non-capsulated
• Fastidious & slow grower
• Microaerobic (needs an atmosphere of reduced O2 (5% o2) and increased CO2 (10% CO2))
  -Thermophilic (42°C) (needs special incubator)
• Filterable

Question 17

What are the species of Campylobacter

Answer 17

• C. jejuni (most common)
• C.coli
• C.fetus
• C.upsaliensis

There are 17 species and 6 subspecies assigned to the genus Campylobacter

Question 18

In whom is the peak incidence of campylobacter associated disease?

Answer 18

Infants and young children
20-40 yrs old adults

Question 19

Who are prone to having severe disease (complications) after infection with Campylobacter?

Answer 19

Patients with hypogammaglobulinemia (low production of antibodies)

Question 20

Which food is most associated with Campylobacter jejuni

Answer 20

Undercooked meats, especially poultry, have been
associated with infection. Commercially raised poultry is nearly always colonized with C. jejuni.

Question 21

Can C. jejuni be isolated from healthy people?

Answer 21

Yes

Question 22

At what age is Campylobacter infection especially common?

Answer 22

During the first 5 years of life

Question 23

How is C. jejuni transmitted?

Answer 23

Through contaminated food, milk, or water; contaminated poultry

Question 24

What is the pathogenesis of Campylobacter?

Answer 24

1. Ingestion of contaminated food or water, especially undercooked poultry, unpasteurized milk, or contaminated produce.
2. Adherence (by adhesins) and invasion (by capsule and flagella) to the epithelial cells lining the gastrointestinal tract, particularly in the jejunum, ileum, and colon.
3. Mucosal damage: Campylobacter infection causes histological damage to the mucosal surfaces of the intestine. The mucosal surface appears ulcerated, edematous, and may exhibit bloody diarrhea. The invasion of the intestinal tissues triggers an inflammatory response, characterized by the infiltration of neutrophils and eosinophils.
4. Toxin production: Campylobacter jejuni produces several endotoxins
5. Immune response: The host immune response plays a crucial role in combating Campylobacter infection. Neutrophils and other immune cells are recruited to the site of infection to eliminate the bacteria. However, the Lipooligosaccharide (LOS) of Campylobacter can evade the immune response and persist in the intestinal mucosa.

Question 25

What type secretion system does Campylobacter have?

Answer 25

Type 4 Secretion System
Usually other bacteria have T3SS

Question 26

What is special about C. fetus?

Answer 26

Campylobacter can cause severe disease, particularly in immunodeficient people. C. fetus can cause bacteremia, diabetes, liver disease, alcoholism, or cancer.
The presence of a capsule-like protein called the S protein contributes to its resistance to complement-mediated killing and enhances its ability to cause bacteremia.

Question 27

What are the virulence factors of Campylobacter?

Answer 27

Cellular components:
> Endotoxin
> Flagellum: Motility: Important for colonization and cell invasion
> Capsule: Facilitates invasion of mucosal epithelial cells
> Lipooligosaccharide: Facilitates immune avoidance and is associated with post infectious complications of certain autoimmune disease
> Adhesins: Mediate attachment to mucosa

Question 28

Which Campylobacter species is reported to have caused human gastrointestinal infection?

Answer 28

•Campylobacter jejuni
•Campylobacter coli

Question 29

Which Campylobacter species is reported to have caused human extra-intestinal infection?

Answer 29

•Campylobacter jejuni
•Campylobacter coli
•Campylobacter fetus

Question 30

Can Campylobacter cause human dental infection?

Answer 30

Yes. Some species (C. concisus, C. curvus) have been reported to have caused human dental infection.

Question 31

What is the incubation period of Campylobacteriosis?

Answer 31

IP: 1-7 days

Question 32

What are the symptoms of Campylobacteriosis?

Answer 32

• Diarrhea (frequently dysenteric)
• Abdominal pain
• Fever
• Headache
• Nausea
• Vomiting

Question 33

What are the complications of Campylobacteriosis?

Answer 33

1) Reactive Arthritis
> Patients with the HLA-B27 phenotype Campylobacter can suffer from reactive arthritis, which is a painful inflammation of the joints which can last several months.

2) Guillain-Barré syndrome (auto-immune disease of the PNS)
> Antigenic cross-reaction between the LPS of Campylobacter and peripheral nerves gangliosides (monosialotetrahexosylganglioside) due to similar carbohydrate structure → a polio-like form of paralysis that can result in respiratory and severe neurological dysfunction or death in a small number of cases.

Begins in lower extremities and ascends bilaterally =
1) Weakness
2) Ataxia (loss of control)
3) Bilateral Paresthesia (numbness) Progressing to Paralysis.

Question 34

What specimen is obtained for the diagnosis of Campylobacter?

Answer 34

Diarrheal stool/rectal swab (has to be processed within 2 hours)

Question 35

Explain the diagnostic steps taken to identify Campylobacter

Answer 35

1) Gram-stain: Stool smear shows seagull shaped rods

2) Culture
a. Filtration: Stool samples are filtered using a 0.45 μm filter to remove larger particles and debris.

b. Culture media: The filtered specimen is then inoculated onto selective culture media, such as Blood Agar Plates (BAP), which contain specific antibiotics to inhibit the growth of competing bacteria.

c. Microaerobic environment: Campylobacter bacteria require a reduced oxygen concentration for optimal growth. Therefore, the plates are incubated in an atmosphere with reduced oxygen (5% O2), increased carbon dioxide (10% CO2), and 85% N2

d. Temperature: Campylobacter species are typically cultured at an elevated temperature of 42°C, which helps to suppress the growth of other bacteria and promote the growth of Campylobacter.

3) Selective media: In addition to the culture conditions mentioned above, selective media specific for Campylobacter, such as Campylobacter Blood Free Selective Agar (CCDA), may also be used to enhance the isolation of Campylobacter colonies.

4) Commercial immunoassay (EIA)
> Not species specific

1. Microplate EIA test: This immunoassay is conducted on a microplate, where the wells are coated with specific antibodies targeting Campylobacter antigens. Stool sample containing Campylobacter antigens will bind to the antibodies. The bound antigens are then detected using enzyme-labeled antibodies, resulting in a color change or signal that can be measured.
2. Lateral flow immunochromatography: This immunoassay format is commonly used in rapid diagnostic tests. A lateral flow strip contains specific antibodies immobilized on different zones. The sample, such as a stool specimen, is applied to the strip, and if Campylobacter antigens are present, they will bind to the antibodies. The antigens and antibody complexes then migrate along the strip through different zones. In the detection zone, immobilized antibodies capture the antigen-antibody complexes, producing a visible signal, such as a colored line.

Question 36

What is the treatment for Campylobacter infections?

Answer 36

> Campylobacter gastroenteritis is typically a self-limited
infection managed by the replacement of lost fluids and
electrolytes
Antibiotic therapy may be used in patients with severe
infections or septicemia: Macrolides/ Tetracycline,
Amoxicillin/clavulanic acid/Imipenem

Question 37

How is campylobacter infection prevented?

Answer 37

• Proper preparation of food ( Chicken and Turkey)
• Avoiding unpasteurized dairy products, and the implementation of safeguards to prevent the contamination of water supplies

Question 38

Name the characteristics of Helicobacter pylori

Answer 38

• Curved gram negative rods
• Very motile —> move in a corkscrew motion
• Microaerophillic— Needs 10% CO2 and 5%
• Unique metabolism (use amino acids and fatty acids rather than carbohydrates to obtain energy)
• Produce Urease
• Prpduce Oxidase
• Produce Catalase
• Growth at 37°C, NOT 25°C or 42°C

Question 39

How is Helicobacter pylori transmitted?

Answer 39

• Fecal/Oral
• Oral/Oral
• Gastric/Oral

> Household transmission-mostly acquired during childhood
Incidence of Helicobacter 📈in Developing countries

Question 40

Pathogenesis of Helicobacter pylori?

Answer 40

Helicobacter pylori synthesizes urease, an enzyme that breaks down urea into ammonia and carbon dioxide. The ammonia generated by urease activity can be damaging to the gastric mucosa, leading to inflammation and contributing to the development of gastritis and peptic ulcers.

The production of ammonia by H. pylori also contributes to its survival in the stomach. Ammonia can neutralize the acidic pH of the stomach, creating a more favorable environment for the bacterium.

Question 41

What are the virulence factors of Helicobacter pylori?

Answer 41

1. Blockage of acid production by a bacterial acid-inhibitory protein, helping the bacterium survive in the stomach’s acidic environment.
2. Neutralization of gastric acids by ammonia produced by bacterial urease activity: H. pylori produces urease, which breaks down urea into ammonia. Ammonia can neutralize gastric acid, creating a more favorable environment for the bacterium to survive.
3. H. pylori is highly motile due to its flagella, allowing it to move through the mucus layer and colonize the gastric epithelium.
4. H. pylori possesses adhesion proteins that help it adhere to and colonize the gastric epithelial cells.
5. H. pylori has lipopolysaccharides (LPS) on its outer membrane that mimic the Lewis blood group antigen. This molecular mimicry helps the bacterium evade the immune system by interfering with the recognition and response of host immune cells.
6. Tissue damage:
   a. Urease products, mucinase, phospholipases: H. pylori produces enzymes such as mucinase and phospholipases that can damage the gastric mucosa, promoting inflammation and tissue injury.b. Vac A: Vacuolation cytotoxin A is a protein produced by H. pylori. When it is taken up by epithelial cells, it induces the formation of vacuoles and can disrupt cellular processes.c. Cag A: Cytotoxin-associated gene A is encoded by a specific gene in some strains of H. pylori. CagA is injected into host epithelial cells via a type IV secretion system. It interferes with cellular pathways, disrupting the cytoskeletal structure and potentially leading to cellular changes.d. PAI: Cag phosphoribosylanthranilate isomerase is part of the pathogenicity island (PAI) of H. pylori. It induces the production of interleukin-8 (IL-8), which recruits neutrophils to the site of infection. Neutrophil activation and enzyme release can contribute to tissue damage and the formation of gastric ulcers.

Question 42

What are common ulcer sites caused by Helicobacter pylori?

Answer 42

Stomach
Small intestine
Large intestine
Esophagus

Question 43

What are the two types of clinical acquisition of Helicobacter pylori?

Answer 43

1) Acute acquisition:
> Nausea, vomiting, and abdominal pain for one week
> Later, gastritis develops

2) Persistent colonization
> Helibacter stays dormant for years after acquisition and person appears asymptomatic
> Can suddenly causes ulcers

Question 44

What are the clinical manifestations of Helicobacter pylori?

Answer 44

• Duodenal ulcer
- More than 90% with duodenal ulcers - carry HP.
- antimicrobial therapy response, eradication of HP - less recurrences

• Gastric ulcer - 50-80% HP

• Gastric carcinoma -HP induces gastritis, gastritis is risk factor for Carcinoma.

• Gastric lymphoma - MALToma: mucosa associated lymphoid tumors, strong association with HP. Stage 1 is cured by antibiotics

Question 45

What is the laboratory diagnosis of Helicobacter pylori?

Answer 45

1) Endoscopy (to see extent of invasiveness) and gastric biopsy
2) Urease detection (urea breath test) !!!
3) Culture on chocolate agar or skirrow medium (blood agar supplanted with antibiotics)
> since Helicobacter is hard to grow like campylobacter
4) Stool Helicobacter antigen test
5) Blood Serology (Chromatography, ELISA)

Question 46

Explain the basis of the urea breath test

Answer 46

Samples of breath air are collected by having
the patient blow into a tube before and 30 min
after ingestion of 14C-labeled urea (converted by urease of Helibacter to 14C-labeled CO2)
It is rapid, noninvasive, for assessing response 4-8w post therapy, expensive but non invasive!!

Question 47

What therapy is used for treatment of Helicobacter pylori?

Answer 47

1. Proton Pump Inhibitors (PPI) therapy: PPIs, such as omeprazole or lansoprazole, are medications that reduce the production of gastric acid, HP, and urease.
2. PPI + Amoxicillin + Clarithromycin or Metronidazole: This is a standard triple therapy regimen for H. pylori eradication.
3. Triple therapy: PPI + Bismuth + Metronidazole + Amoxicillin: (BEST) Bismuth has antimicrobial and anti-inflammatory properties, aiding in H. pylori eradication.