Miscellaneous Fastidious GNB, C/B and GNC

Question 1

Haemophilus - Description

Answer 1

General Characteristics:

Genus of Pasteurellaceae family

Some species moved to Aggregatibacter

Small, pleomorphic, non-motile, non-sporing Gram-negative rods or cocco-bacilli

Aerobic & facultatively anaerobic

Growth enhanced by 5–10% CO₂

Oxidase & catalase reactions vary

Requires X (hemin) and/or V (NAD) factors for growth

Question 2

Haemophilus - Clinical Significance

Answer 2

H. influenzae:

Colonises URT of healthy individuals

Hib (Type b): Pre-1989, leading cause of bacterial meningitis in 6m–5yr children

Non-typable (NTHI) colonisation ~80%

Transmission: direct contact, secretions, aerosols

H. ducreyi:

Sexually transmitted (chancroid)

Requires X factor only

No animal reservoir

H. parainfluenzae, H. parahaemolyticus:

Normal URT flora

Requires V factor only

Question 3

Haemophilus - Identification

Answer 3

Colonial Morphology:

Growth enhanced by CO₂

Chocolate agar: small, round, convex colonies

Some species haemolytic

Cellular Morphology:

Small Gram-negative rods or cocco-bacilli

Tend to grow in pairs

In clinical samples, may appear filamentous on rich media

Question 4

Haemophilus - Growth Factor Requirements

Answer 4

General Growth Requirements:

Most species require blood factors for growth

Chocolate agar (Choc) used – heating releases factors from RBCs

Growth occurs only where both X & V factors have diffused

Factor X (Haeme, Haemin, Haematin):

Essential for catalase, peroxidase, Cytochrome C (ETC)

Diffuses from intact RBCs (available on Blood Agar (BA))

Factor V (NAD - Nicotinamide Adenine Dinucleotide):

Required for oxidation-reduction processes in metabolism

Does NOT diffuse from intact RBCs (no growth on BA if needed)

Culture on Nutrient Agar:

Growth only occurs if X & V factors are provided externally

Question 5

Haemophilus - Satellitism

Answer 5

Definition:

Satellitism refers to small pinpoint colonies of Haemophilus growing around other bacteria that provide V factor (NAD)

Growth on Sheep Blood Agar (SBA):

Haemophilus will only grow if V factor is supplied by another organism (e.g., Staphylococcus aureus)

Sheep blood differs from horse/rabbit blood:

Contains enzymes that hydrolyse V factor, preventing its availability

Question 6

Haemophilus - Sample Collection & Culture

Answer 6

Respiratory Secretions & CSF:

Must be cultured on chocolate agar

H. ducreyi:

Grows on Mueller-Hinton (MH) agar with 5% sheep blood in CO₂

CSF:

In untreated patients, contains 2 × 10⁷ bacteria/mL

~80% visible via microscopy

Gram-Stained Sputum:

Shows small, pleomorphic, Gram-negative coccobacilli

5 × 10⁸ organisms/mL of sputum

Blood Cultures:

10–15% positive in pneumonia cases

~50% positive in meningitis cases

Question 7

Bordetella - Description

Answer 7

Tiny, Gram-negative coccobacilli on primary isolation

Contains 9 closely related species, including:

B. pertussis (whooping cough)

B. parapertussis (milder whooping cough)

B. bronchiseptica (affects animals & immunocompromised humans)

B. avium, B. hinzii, B. holmesii, B. trematum, B. ansorpi, B. petrii

Question 8

Bordetella - Epidemiology

Answer 8

B. bronchiseptica: Normal flora of the respiratory tract in animals (causes disease in pigs & dogs)

B. pertussis survives outside the body for days, transmitted via contaminated objects

B. pertussis & B. parapertussis:

No animal reservoir

Infect ciliated epithelium of human respiratory tract

Highly contagious, spreads via aerosol droplets from coughing

Question 9

Bordetella pertussis - Clinical Significance

Answer 9

Acute respiratory illness

Early symptoms: Mild cough, sneezing, coryza, slight fever

Progression:

Severe paroxysmal coughing fits

Characteristic “whooping” sound when inhaling

Post-tussive vomiting, cyanosis, apnea (life-threatening)

“100-day cough” (prolonged duration)

Communicability: 30–90%

Vaccine available (older children/adults can act as reservoirs)

Question 10

Bordetella - Culture & Transport

Answer 10

Samples:

Nasopharyngeal swabs, aspirates, tracheal aspirates, throat swabs

Transport Medium:

Half-strength charcoal blood agar (Regan-Lowe medium)

Minimize transport time

Culture Isolation:

B. pertussis & B. parapertussis require charcoal or 20% blood in agar

Other Bordetella species grow on Blood Agar (BA), MacConkey (MAC), Chocolate (Choc) agar

None require X & V factors

Question 11

Bordetella - Key Differential Characteristics

Answer 11

Extremely small, strictly aerobic, Gram-negative, non-motile coccobacillus

Species Oxidase Urease Motility
B. pertussis +ve -ve Non-motile
B. parapertussis -ve +ve Non-motile
B. bronchiseptica +ve +ve Motile

Question 12

Brucella - Overview

Answer 12

Gram-negative coccobacilli

Non-spore-forming & non-motile

Aerobic, but may require added CO₂

Zoonotic pathogen – transmitted from animals to humans

Humans are accidental hosts

Six recognized species (may be reduced to B. melitensis + biovars)

Named after their primary animal hosts:

B. abortus (cattle) ()

B. melitensis (sheep & goats) (*)**

B. suis (pigs) ()**

B. canis (dogs, esp. beagles) (*)

B. ovis (sheep)

B. neotomae (rats)

Question 13

Brucella - Transmission

Answer 13

Three main transmission routes:

Direct contact with infected animal tissue

Ingestion of contaminated meat or dairy products

Inhalation of aerosolized organisms

Question 14

Brucella - Clinical Significance (Brucellosis)

Answer 14

Enters through mucous membranes

Spreads via macrophages to lymph nodes & spleen

Can also spread to uterus, placenta, prostate gland, and internal organs

Prefers organs rich in erythritol (sugar used instead of glucose)

Found in animal tissues: breast, uterus, placenta

Causes “contagious abortion” in animals

Humans lack erythritol, so no specific tissue localization

Symptoms:

Fever, GI upset, arthritis, respiratory tract symptoms

Can cause abortions & sterility

Mild or asymptomatic disease in natural hosts

Question 15

Brucella - Isolation & Culture

Answer 15

Primary specimens:

Blood or bone marrow

Slow-growing organism (takes 4-6 days at 37°C)

Growth media:

Blood Agar (BA), Chocolate Agar (Choc), Modified Thayer-Martin (MTM)

Colony morphology:

Small, smooth, non-haemolytic colonies

Key Identification Features:

Intracellular, small coccobacilli

Non-motile

Strict aerobes, prefer CO₂

Catalase & oxidase positive

Question 16

Bartonella - Overview & Clinical Significance

Answer 16

Only genus in the Bartonellaceae family

Facultative intracellular parasites – survive in erythrocytes

Transmitted by arthropod vectors

Infects both healthy individuals & opportunistically in immunocompromised (IC) patients

Key Bartonella species & diseases:

B. henselae → Cat scratch disease (immunocompetent)

Bacillary angiomatosis (IC patients, spread by cats/fleas)

B. bacilliformis → Carrión’s disease (humans, sandflies)

B. quintana → Trench fever (humans, body lice)

Infection occurrence depends on the geographic distribution of host & vector

Question 17

Bartonella - Diagnosis

Answer 17

Grows slowly on blood agar with 5-10% CO₂

Culture is not a sensitive or timely method (may take weeks)

Most cases diagnosed by serology

Question 18

Campylobacter & Helicobacter - Overview

Answer 18

Originally part of the same phylogenetic group

Family: Campylobacteriaceae

Campylobacter:

31 species, 13 subspecies, 12 cause human disease

Main species: C. jejuni, C. coli

Helicobacter:

Family not yet named

Colonizes mucosal surfaces of GI & reproductive tracts

Spiral shape + polar flagella → rapid corkscrew motility

Question 19

Campylobacter - Background

Answer 19

First linked to abortion in cattle/sheep (early 20th century)

Initially called Vibrio, later reclassified as Campylobacter (1973)

Common in the GI tract of animals, especially poultry

Major cause of bacterial gastroenteritis worldwide

Thermophilic species (C. jejuni, C. coli) grow at 42°C

Question 20

Campylobacter - Epidemiology

Answer 20

Major zoonotic cause of gastroenteritis & foodborne disease

High-income countries: Symptomatic in all ages

Low-income countries: Mostly affects children <5 years

Animals serve as reservoirs & contamination sources (food, water, environment)

Direct & indirect animal contact is a risk factor, especially for children

Question 21

Campylobacter - Description

Answer 21

Small, curved Gram-negative rods

Motile via polar flagella → Corkscrew motility

Microaerophilic (5% O₂, 10% CO₂, 85% N₂)

Thermophilic species grow better at 42°C

Requires special isolation media (CCDA)

Question 22

Campylobacter - Culture

Answer 22

Difficult to culture from stool samples

Slow growing (48-72 hrs)

Selective media required:

SBA + vancomycin, cephalothin, trimethoprim (inhibits normal gut flora)

Growth at 42°C

Enrichment broth (Campy broth) before plating

CCDA (Charcoal cefoperazone deoxycholate agar)

C. jejuni → grey, moist, flat, spreading colonies

Question 23

Campylobacter - Colonial Morphology

Answer 23

Grey, flat, glossy colonies spreading along streak lines

Resemble droplets of fluid on moist agar

As agar dries, colonies become low & convex

C. jejuni → metallic sheen over time

C. coli → more convex, shiny colonies

Question 24

Hippurate Test (C. jejuni vs. C. coli)

Answer 24

C. jejuni = Hippurate positive (+)

C. coli = Hippurate negative (-)

Hippuricase hydrolyzes hippurate → Produces glycine

Detect glycine → Blue/purple color

Question 25

Campylobacter - Identification

Answer 25

Gram stain: "Seagull" shape Direct Gram stain of feces + RBCs & polymorphs Stain poorly with safranin → Leave on 2-3 min or use carbol fuchsin Darting motility (under wet mount) Hippurate positive + Oxidase positive

Question 26

Antibiotic Susceptibility for Identification

Answer 26

Cephalothin resistance = C. jejuni Nalidixic acid sensitivity = C. jejuni Fluoroquinolone resistance increasing

Question 27

Helicobacter pylori - Description

Answer 27

A Gram-negative, spiral-shaped rod Highly motile with 4–6 sheathed flagella at both poles Produces large amounts of urease & catalase Can survive stomach acidity by burrowing into gastric mucosa

Question 27

Helicobacter pylori - Clinical Significance

Answer 27

Associated with peptic ulcer disease, gastric ulcers, MALT lymphoma, and gastric cancer 90% of duodenal ulcer patients are infected with H. pylori Chronic superficial gastritis can lead to ulcers due to increased acid secretion Most infections (80–90%) are asymptomatic, but 10–20% develop severe gastric disease

Question 28

Helicobacter pylori - Risk Factors & Prevalence

Answer 28

Higher prevalence in low socioeconomic childhood environments (up to 90%) In Western countries: 50% of people over 60 are infected 20% of people under 40 are infected

Question 29

Helicobacter pylori - Cultural Characteristics

Answer 29

Specimen: Gastric biopsy (from endoscopy) Culture Conditions: Microaerophilic atmosphere Non-selective agar with 5–10% human blood Incubation for 7–10 days (colonies appear in 3–4 days) Colony Morphology: Small, round, translucent colonies

Question 30

Helicobacter pylori - Identification Methods

Answer 30

Gram Stain: Carbol fuchsin counterstain for 2–3 min 🔬 Histology: H&E, Giemsa, Silver stains on tissue sections 🔬 PCR: Detects H. pylori and clarithromycin resistance

Question 31

Rapid Urease Test (RUT) for H. pylori

Answer 31

1️⃣ Biopsy placed in a urea-containing medium with phenol red (pH indicator) 2️⃣ If H. pylori is present, urease converts urea to ammonia + CO₂ 3️⃣ Increased pH → Color change from yellow to pink ✅ Rapid results (minutes to hours) ⚠️ False negatives if the patient has taken antibiotics

Question 32

Pathogenic Neisseria Species

Answer 32

Neisseria meningitidis Colonizes the oropharynx & nasopharynx Opportunistic pathogen Neisseria gonorrhoeae Strict human pathogen (not part of normal flora) Causes gonorrhoea and systemic infections

Question 33

Neisseria Cellular Morphology

Answer 33

Gram-negative, oval diplococci Occur in pairs with opposed sides flat/slightly concaved "Coffee bean" or "kidney bean" appearance Often intracellular within PMNs in clinical specimens N. gonorrhoeae → urethral discharge N. meningitidis → CSF

Question 34

Neisseria gonorrhoeae - Clinical Overview

Answer 34

Sexually transmitted infection (STI) (2nd most common after Chlamydia) Localised infection at initial exposure site Diseases include: Gonorrhoea (urethritis, cervicitis) Pelvic inflammatory disease (PID, 10-20%) Disseminated gonococcal infection (DGI, 1-3%) Conjunctival infections Humans only (strict pathogen) 15-24 years age group Core transmitters → High-risk sexual networks

Question 35

Neisseria gonorrhoeae - Culture Requirements

Answer 35

Fastidious (difficult to grow) Requires enriched media: Chocolate agar Thayer-Martin agar / Modified TM / Martin-Lewis agar Selective media with antibiotics: Vancomycin (inhibits Gram-positives) Colistin (inhibits Gram-negatives) Nystatin/Amphotericin B (inhibits fungi) Trimethoprim (inhibits Proteus) Incubation conditions: 5% CO₂, high humidity, 37°C, 24-72 hrs

Question 36

Neisseria gonorrhoeae - Identification

Answer 36

Gram-negative diplococci with PMNs (urethral discharge smear) Colonial morphology on selective media: 0.5mm, raised, glistening, grey-white colonies Biochemical tests: Oxidase +, Catalase + Glucose fermentation + (maltose -) Does not survive long outside the host → requires enriched media & CO₂ Small colony size (0.5mm) → slow-growing, fastidious nature

Question 37

Neisseria meningitidis - Clinical Overview

Answer 37

Colonizes the oropharynx & nasopharynx (8-40% carriage rate) Spread via aerosolized respiratory droplets Highest incidence: Infants (6 months – 5 years) Young adults Community-acquired meningitis (2nd most common cause in adults) Meningococcal septicemia (can lead to DIC, shock) Meningitis outbreaks linked to capsular serogroups A, B, C (90% cases)

Question 38

Neisseria meningitidis - Culture & Identification

Answer 38

Gram stain of CSF (highly sensitive & specific) Culture on Blood Agar (BA) & Chocolate Agar (Choc): 3-10% CO₂, high humidity, 37°C Colonial morphology: Larger than gonococci (1-2mm) Round, convex, smooth, glistening grey colonies Carbohydrate Utilization Test: Species Glucose Maltose Lactose N. gonorrhoeae + - - N. meningitidis + + -

Question 39

Clinical Significance of Moraxella catarrhalis

Answer 39

Human-only opportunistic pathogen (respiratory mucosa) Frequently colonizes the nasopharynx asymptomatically Causes respiratory tract & adjacent infections, including: Otitis media (OM) in children Sinusitis Bronchopulmonary infections Acute exacerbations of COPD in adults Systemic disease in elderly & immunocompromised

Question 40

Cellular Morphology of M. catarrhalis

Answer 40

Gram-negative, short, plump cocci Aerobic Resists Gram stain destaining Forms pairs (diplococci) or tetrads Adjacent sides flattened (like Neisseria)

Question 42

Culture Characteristics of M. catarrhalis

Answer 42

Grows on Blood Agar (BA) & Chocolate Agar (Choc) Not fastidious (unlike Neisseria) Incubation: 5-10% CO₂, 24 hrs Colony morphology: Nonhemolytic, round, opaque, convex, greyish-white "Hockey puck" colonies (move intact across agar when pushed) Large (~2mm) sticky colonies on Choc & BA

Question 43

Biochemical Identification of M. catarrhalis

Answer 43

Oxidase + Catalase + Non-motile, aerobic Carbohydrate fermentation: Asaccharolytic (Negative for glucose, maltose, lactose, sucrose) Differentiation from Neisseria: Hydrolyzes indoxyl butyrate (Neisseria does not) Indoxyl butyrate test: Produces blue-green color if positive