Untitled Quiz

Gram-Negative Coccobacilli

• Gram-negative bacteria with a rod-like or slightly oval shape
• Clinically significant microorganisms frequently isolated in the body
• Bacteria do not grow on MacConkey agar (MAC)
• Important examples include Haemophilus influenzae, related organisms, and bacterial virulence

Haemophilus

• Facultative anaerobes ("blood-lovers")
• Most species are part of the normal flora of the upper respiratory tract
• Pathogenic species include:
  • Haemophilus influenzae
  • H. influenzae biogroup aegyptius
  • H. ducreyi (genital tract pathogen)
• Optimal growth for most species is in 5-10% CO2 at 35 +/- 2 degrees Celsius
• H. aphrophilus requires CO2

Haemophilus Nutritional Requirements

• Fastidious bacteria
• Do not grow on routine media (BAP, TSA, MAC)
• Require accessory growth factors found in red blood cells (RBCs):
  • X factor (hemin/hematin)
  • V factor (nicotine adenine dinucleotide or NAD) or both
• Species with "para" in their name require only V factor
• Media with horse or rabbit blood provides necessary growth factors
• Sheep blood contains X-factor readily available. V factor must be obtained

Haemophilus Growth on BAP

• Additional V factor source can be added to routine BAP
• Fastidious organisms grow as "satellites" surrounding other bacteria releasing the necessary growth factors or toxic products
• S. aureus, S. pneumoniae, and Neisseria species naturally produce V factor

Haemophilus influenzae -- Virulence factors

• Only in typeable strains
• Six types based on polysaccharide capsules—a, b, c, d, e, f
• Type B most common before vaccination
• Implicated in meningitis in children
• Can be typed as needed
• IgA proteases
• Destroys secretory immunoglobulin A (sIgA)
• Adherence via fimbriae and structures
• Damages localized areas in non-typeable strains
• Outer membrane proteins and lipopolysaccharide (LPS)

Haemophilus influenzae -- Infections

• Most common in children
• Organism enters the body through nasopharynx (colonizes) may remain transiently
• May enter bloodstream causing invasive infections
• Two patterns:
  • Invasive disease (e.g., bacteremia, septicemia, meningitis, arthritis, epiglottitis*, tracheitis, and pneumonia) caused by encapsulated strains
  • Localized infection caused by non-encapsulated strains

Haemophilus influenzae -- Specimens

• Specimens of choice: -Blood -CSF -Sputum -Conjunctival swabs
• Transport specimens at room temperature without delay because organisms die rapidly

Haemophilus influenzae -- Colonial Morphology

• On CHOC agar:
  • Colonies are large, translucent, and moist
  • Distinct pungent odor (mousey or bleach-like)
  • Encapsulated strain colonies appear mucoid
• On BAP:
  • No growth with no staph streak
  • With staph streak - Satelliting colonies
• Morphology will help in identification

Haemophilus influenzae -- Cellular Morphology

• Pleomorphic appearance (various shapes)
• Tiny gram-negative coccobacilli (Coccobacilli)
• Clear non-staining areas (halos) may be seen (capsules)
• Faint staining; Ensure microscope is clean

Haemophilus influenzae -- ID

• Tiny gram-negative pleomorphic coccobacilli on CHOC agar
• Grows only on CHOC agar, not MAC
• BAP with staph streak – satellite growth
• Oxidase-positive
• X and V Factor Test: -Growth around X + V disk only -No growth around X or V disk
• Porphyrin test (ALA): negative
• Catalase-positive
• Latex Agglutination and Coagglutination kits to detect Hib antigen
• Chromogenic tests – API NH

Haemophilus influenzae -- X and V Factor

• Purpose: Identify growth requirements for the speciation
• Principle: Clear agar missing X and V factor to inoculated with the target organism
• Nutrients (X and V factor) added separately
• Incubate and observe for pattern of growth (growth around X or V indicated positive)

Haemophilus influenzae -- ALA test

• Purpose: Differentiation of Haemophilus spp.
• Principle: determines if isolate requires X-factor
  • Organisms needing X factor cannot convert aminolevulinic acid (ALA) to hemin.
  • Organisms producing their own hemin can.
• Tube of ALA or a disk inoculated with organism
• View under UV light to observe for fluorescence for positive result
• Use the appropriate quality controls

Haemophilus influenzae -- Treatment

• More than 35% of strains produce Beta-lactamases (resistance to ampicillin) - common in meningitis and bacteremia
• Testing for Beta-lactamase activity
• Recommended treatment (beta-lactamase positive):
  • Cefotaxime, ceftriaxone, and cefuroxime
• Alternate treatment:
  • Trimethoprim/Sulfamethoxazole (SXT), imipenem, ciprofloxacin (with ampicillin).

Haemophilus influenzae -- Vaccination

• Hib vaccination recommended for children under 5 years
• Children given multiple shots
• Pre-Vaccination Era -Leading cause of invasive disease in children -Common cause of acute bacterial meningitis in children until mid-1980s

Haemophilus influenzae biogroup aegyptus

• Subgroup of H. influenzae, closely related
• Very difficult to differentiate
• Infections: Acute, contagious conjunctivitis (pink eye)
• Brazilian purpuric fever
• Sudden onset high fever, rash, septicemia, shock, vascular damage
• Mortality rate as high as 70% within 48 hours
• Small outbreaks in South America

Haemophilus ducreyi -- Infection

• NOT normal flora, genital ulcer disease, chancroid
• Highly contagious sexually transmitted disease
• Painful, purulent ulcers, or buboes (swollen lymph nodes)
• Men have more symptoms related to inguinal tenderness and genital lesions

Haemophilus ducreyi -- Cellular Morphology

• Tiny GNCB (gram-negative coccobacilli)
• Arranged as "school of fish"

Haemophilus ducreyi -- Culture and ID

• Requires enriched CHOC
• Specimens inoculated immediately

Haemophilus species (other than H. influenza, and H. ducreyi)

• Typically non-pathogenic but associated with infections like (endo)carditis, Respiratory tract infections, brain abscesses, bacteremia, urethritis, and pneumonia

HACEK Group

• Gram-negative bacilli
• Do not grow on MacConkey agar
• Fastidious nutritional requirements
• Require an increased CO2 (5%-10%) environment
• Significant cause of endocarditis
• Usual oral cavity flora
• Opportunistic in immunocompromised hosts

Capnocytophaga

• Group of fastidious, facultatively anaerobic, gram-negative bacilli
• Normal flora in oral cavity
• Infections in neutropenic patients -Oral ulcers -Soft tissue infections, peritonitis, and endocarditis

Pasteurella -- General Characteristics

• Many species, most clinically relevant: Pasteurella multocida
• Has similar characteristics to Haemophilus
• Infections: Causes pasteurellosis
• Disease acquired from infected animals or products
• Zoonotic disease
• Animal bites, often cat bites

Pasteurella -- Infections

• Common presentation: soft tissue infection
• Injury can be aggressive, with skin manifestations typically appearing within 24 hours
• Wounds exhibit rapidly progressive soft tissue inflammation
• Soft tissue infections can progress to deep-tissue infections (Septic arthritis, osteomyelitis, tenosynovitis)
• Rarer presentation: -Systemic infections (endocarditis, septicemia, meningitis, pneumonia)

Pasteurella -- Cellular Morphology

• Can easily be mistaken with Haemophilus
• Gram-negative coccobacilli
• Ovoid, filamentous, or bacilli
• Bipolar staining (looks like safety pins)
• End of bacillus retains more stain

Pasteurella -- Colonial Morphology

• Not fastidious - Grows on BAP and CHOC
• Growth on BAP helps differentiates from Haemophilus
• Tends to exhibit a "musty" odor similar to wet dog smell
• Nonhemolytic, grey colonies
• Older colonies—mucoid and green/brown halo.

Pasteurella -- Diagnosis

• Nonmotile
• Catalase positive, Oxidase positive (most isolates)
• Nitrate positive, Indole Positive
• Ornithine Positive
• Glucose fermentation -Weak acid production
• Urease negative

Other Knowledge Checks

• Specific characteristics to distinguish between Haemophilus and Pasteurella
• Organisms causing diseases like soft chancres, or brazilian purpuric fever
• Identification of organisms not in the HACEK group
• Organism commonly associated with animal bites causing soft tissue infections