Brucellosis and Haemophilus Influenzae Quiz

Haemophilus and Other Fastidious Gram-Negative Bacilli

• These bacteria are often difficult to grow in standard laboratory conditions.
• They belong to the family Pasteurellaceae, with Haemophilus being a significant genus.
•  Haemophilus species are gram-negative coccobacilli or rods.
• They are nonmotile and able to utilise aerobic to facultatively anaerobic conditions.
• Most Haemophilus species require growth factors (X and V factors)

Gram Stain of Haemophilus

• Haemophilus influenzae and H. parainfluenzae are commensal bacteria in the upper respiratory tract of humans.
• H. ducreyi is found in the genital tract and is transmitted sexually.
• A gram stain of Haemophilus will show small, gram-negative bacilli.

Haemophilus species

• Gram-negative pleomorphic coccobacilli or rods
• Smears reveal coccobacilli and occasionally long filamentous rods from colony growth.
• These bacilli are nonmotile, oxidase positive, catalyse positive, and often reduce nitrate.
• Obligate parasites of the mucous membranes.
• Ten species involved in human infection (H. influenzae, H. haemolyticus, H. aegyptius, H. influenzae biotype aegyptius, and H. ducreyi).
• Require X factor (hemin, hematin) and V factor (nicotinamide-adenine dinucleotide [NAD])

Haemophilus species (cont'd)

• Some Haemophilus species only need V factor.
• H. hemolyticus species and sometimes H. ducreyi grow on sheep blood agar but not V factor.

Growth patterns

• Satellite growth is a typical characteristic of fastidious organisms like Haemophilus.
• Bacteria that release the necessary growth factors or breakdown toxic products surrounds the fastidious organisms.
• Staphylococcus aureus and Streptococcus pneumoniae often exhibit this behaviour.

Growth patterns (Cont'd)

• H. aphrophilus and H. ducreyi are notable exceptions to the satellitism pattern

Haemophilus influenzae: Historical Perspective

• Isolated from pandemic influenza victims, but not usually the primary agent
• Often found in secondary infections.

Virulence Factors

• Capsule: Serotypes a-f, important antiphagocytic factor
• IgA protease: Cleaves IgA on mucous membranes
• Outer membrane proteins and lipopolysaccharide (LPS): Not well defined, but antibodies may confer some protection, and LPS can paralyze cilia, preventing lung clearance
• Adherence: Pili and other structures

Capsule (cont'd)

• Classified by capsule serotype, containing ribitol and phosphate.
• Antiphagocytic and anticomplement activity
• H. influenzae serotype b (Hib) is a primary cause of the disease in children, often involving bacteremia.

Capsule (Cont'd)

• Nontypeable strains lack capsules and are primarily associated with localized respiratory tract infections like pneumonia, otitis media, and sinusitis

Pasteurellaceae - Haemophilus - Antigenic Structure

• Most Haemophilus influenzae strains are covered by a polysaccharide capsule.
• Six antigenic serotypes are known (A to F).
• H. influenzae type B is a major cause of invasive disease.

Pasteurellaceae - Haemophilus - Virulence Factors

• The primary virulence factor for H. influenzae type B is its antiphagocytic polysaccharide capsule (PRP).
• A PRP vaccine administered at two months of age greatly reduces incidents of meningitis and epiglottitis.

Pasteurallaceae - Haemophilus - Diseases

• H. influenzae type B is responsible for meningitis, epiglottitis, septic arthritis, cellulitis, and conjunctivitis primarily in children
• It can cause lower respiratory tract disease.
• H. ducreyi causes genital ulcers known as chancroid.

Types of Haemophilus Infection

• Types of Haemophilus infection are categorized as invasive (mainly in children, leading to meningitis, cellulitis, epiglottitis, and conjunctivitis), and unencapsulated (largely in children, resulting in otitis media) versus encapsuled (mostly in adults, resulting in pneumonia, bronchitis, and sinusitis).

Clinical manifestations of Haemophilus influenzae

• Hib strains cause meningitis, typically in infants and children (age 3 months to 6 years).
• Epiglottitis (inflammation of the epiglottis causing airway obstruction) is common in children (age 2-4 years).
• Bacterial tracheitis, a life-threatening disease, is prevalent in young children.
• Cellulitis, indicated by swelling, pain, and reddish blue inflamed areas, affects children younger than 2 years old
• Other infections such as acute pharyngitis and pneumonia commonly occur in children approximately one year old.
• Nonencapsulated strains frequently cause otitis media, bronchitis/sinusitis, and other infections.

Haemophilus aegyptius

• H. aegyptius, along with the H. influenzae biogroup aegyptius, can cause conjunctivitis and is notably widespread in tropical climates
•  These infections can manifest as recurrent conjunctivitis, high fever, vomiting, and severe bleeding or bruising, sometimes associated with a high mortality rate.

Haemophilus ducreyi

• A sexually transmitted infection that results in chancroid.
• Infection has an incubation period of 4-14 days.
• Characterised by painful genital and perianal lesions with irregular edges.
• Lymph node swelling (buboes) is common.
• Lesions are characterized by a red papule (characteristic of hungry hyperemia), progressing to a pustule, ulcer, and then lymphadenopathy and/or buboes

Miscellaneous Haemophilus species

• H. parainfluenzae: Endocarditis, bite-wound infections
• H. aphrophilus: Endocarditis, bite wound infections

Haemophilus parainfluenzae

• Normal inhabitant in the respiratory tract
• Zoonotic disease of swine (“hog influenza”)
• Found in both upper respiratory and gastrointestinal tracts
• Also cause bacterial endocarditis and pneumonia, often occurring in female genital tract infections

Specimen Processing

• Haemophilus species die rapidly, requiring specimen processing within 10 minutes to maximize recovery rates
• H. ducreyi specimens should be cleaned carefully with sterile saline before being collected from base of the ulcer using a sterile swab
• H. Influenzae specimens are most effectively collected via a swab of the nasopharynx.

Specimen processing (cont'd)

• H. aegyptius specimens should be collected in a chocolate agar supplemented with 1% isovitaleX
• H. ducreyi specimens should be collected and processed in a GC agar supplemented with hemoglobin, FBS, and vancomycin to reduce contamination by other genital flora.
• Haemophilus specimens require incubation in a capnophilic atmosphere with a humidity chamber.

Culturing Haemophilus

• Besides chocolate agar, BAP (blood agar plate) is used to cultivate non-V factor-requiring Haemophilus species, such as H. aphrophilus
• MacConkey agar can also be used for cultivation.
• Colonies should be translucent, moist, smooth, convex, with a mousy or bleach-like odor.
• Encapsulated strains tend to display a mucoid appearance.

Culturing Haemophilus (Cont'd)

• H. influenzae colonies appear as Gram-negative coccobacilli or small rods.
• H. ducreyi colonies appear as coccobacilli in "school of fish" arrangements.

Laboratory Identification

• Colonies are collected from isolation.
• Colonies are placed into nutrient/broth and mixed.
• Nutrient/broth is placed on a nutrient agar plate (or Mueller-Hinton plate) and X and V strips are added.
• Plates are incubated at 35-37°C in 5-10% CO2 for 18-24 hours.
• Plates are read after incubation.

Organism Requiring X and V Factors

• Identify based on growth around X and V factors on the plate.

Organism Requiring V Factor Only

• Identify based on the presence of V factor, but absence of X factor

Organism Requiring X Factor Only

• Identify based on the presence of X factor, but absence of V factor

Porphyrin Test

• If bacteria do not require X factor, ALA is converted to porphyrins and protoporphyrins.
• Kovacs reagent is used to detect reddish-orange fluorescence under the appropriate UV lamp in the lower part of the tube.

Differential Haemophilus speciesTests

•  A table to differentiate various Haemophilus, Aggregatibacter, and Capnocytophaga species.

Differential Haemophilus species Tests (Cont'd)

•  Another table to differentiate Haemophilus biogroups based on biochemical tests, including serotype specific groupings.

Treatment

• Cefotaxime or ceftriaxone is the recommended treatment for most Haemophilus infections.
• Alternative treatments such as trimethoprim-sulfamethoxazole, imipenem, ciprofloxacin, and chloramphenicol are available for varying infections..
• Erythromycin or similar medications are effective for H. ducreyi infections.

HACEK Group General Characteristics

• Gram-negative bacilli
• Require elevated CO2 (5-10%) levels
• Significant cause of endocarditis
• Often found as normal flora in the oral cavity.
• Pathogens in bite wounds, sometimes cause septicemia and subacute endocarditis.
•  Opportunistic in immunocompromised hosts

Aggregatibacter aphrophilus

• Previously classified as a Haemophilus species, it's known for now being a part of the HACEK group (e.g. a gram-negative bacillus).
• Derived from the Greek "foam-loving," suggesting a preferred habitat in similar environments.

Aggregatibacter actinomycetemcomitans

• Primarily recognized as an animal pathogen, often found in the human oral cavity.
•  Nonmotile gram-negative coccobacilli.
• The culture of this organism takes 24–48 hours, and colonies exhibit a unique star-like shape in their centre.
•  This organism is also sensitive to serum based substances such as lactose and sucrose.

Aggregatibacter actinomycetemcomitans (Cont'd)

• Positive for catalase and oxidase tests.
• Not able to grow on MacConkey agar.
• Negative for urease, indole, esculin, and citrate degradation.

Cardiobacterium hominis

• Gram-negative pleomorphic, nonmotile bacillus

Cardiobacterium hominis (Cont'd)

• Frequently implicated in endocarditis.
• Resistant to numerous antibiotics and often requires the replacement of valves in these patients.
• Grows well in blood and chocolate agar and is not as successful in MacConkey agar.
• May show “rosette” appearance in the gram-stain.

Eikenella corrodens

• Normal flora in the human oral and bowel cavities often associated with bite wounds.
• Gram-negative, nonmotile coccobacilli with commonly a bleach-like odor

Eikenella corrodens (Cont'd)

• Positive for oxidase and lysine decarboxylation but negative for catalase, ornithine decarboxylation, and arginine decarboxylation.

Kingella Species

• Gram-negative coccobacilli or short rods frequently appearing as pairs or chains
• Non-motile but can sometimes exhibit twitching motility
• Oxidase and catalase negative
• Able to ferment a variety of sugars

Kingella Species (Cont'd)

• Kingella dentrificans: Colonies that do not pit on Thayer Martin medium, may resemble N. gonorrhoea, often with square-ended rods.

Kingella Species (Cont'd)

• Kingella kingae: Isolates from children under 5 years old are typically negative for sucrose and glucose positive.

Capnocytophaga

• Previously categorized as DF-1 and DF-2 (dysgonic fermenters), it now belongs to a separate group.
• Gram-negative bacilli sometimes showing fusiform/elongated shapes.
• Facultative anaerobes, but usually requiring CO2 for optimal growth.
• Commonly associated with septicemia in humans versus typical endocarditis.

Francisella species

• Gram-negative coccobacillus
• Facultative intracellular parasite commonly found in zoonotic infections (e.g., tularemia)
• Transmitted by ticks, rabbits, rodents, and mosquitos, among other animal vectors

Francisella Species(Cont'd)

• Four subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, and F. tularensis subsp. Novicida.
• F. tularensis subsp. tularensis is typically more virulent

Francisella tularensis

• Aerobic bacillus
• Etiologic agent for tularemia (granular fever, rabbit fever, tick fever)
• Frequently spread by direct contact with animals (e.g., rodents) or arthropods (e.g., ticks).

Legionella Species

• Legionella pneumophila: etiologic agent for Legionnaires' disease and Pontiac fever
• Occurs in 14 serogroups that are mainly found in environmental water sources (e.g., heating towers, air conditioning systems).
• Can also be found in respiratory systems that pass through other people.

Legionella species (Cont'd)

• Infection can range from asymptomatic illness to mortality.

Legionella species (Cont'd)

• Diagnosis can involve culture, testing for antigen in the urine, or using direct fluorescent antibody staining

Legionella

• Culture involves using BCYE media under specific atmospheric conditions (e.g. aerobic 35–37°C at least 7 days).

Legionella - Taxonomy

• Legionella includes over 30 species.
• Legionella pneumophilia is associated with human disease.
• Recognized from Legionnaires infected in Philadelphia during the Legion convention in 1976.

Legionella - Incidence

• The incidence of various Legionella species infections.
• Specifically, the frequency of Legionella pneumophilia serotype 1, 6, other pneumophilia, micdadei, bozemanii, dumoffii, gormanii, longbeachae, all others

Legionella - Habitat

• Found in water sources including heating towers, air conditioners, and plumbing.
• Also present in soil.

Legionella - Virulence factors

• Predisposition for infection is found in several underlying conditions, like smoking, chronic lung diseases, alcoholism, immunosuppressive therapy, and advanced age.
• Intracellular parasites that multiply in alveolar macrophages and monocytes.

Legionnaires' Disease

• Sporadic or epidemic, usually originating from air conditioners
• Can be present in nosocomial infections in immunocompromised patients.
• One major cause of community acquired pneumonia, alongside streptococcus pneumoniae, Mycoplasma pneumoniae, and Chlamydia pneumoniae.

Legionnaires' Disease (Cont'd)

• Incubation period ranges from 2 to 10 days, often accompanied by nonproductive cough, fever, headache, and myalgia.
• Typically results in bloody or purulent sputum.
• Dissemination via circulatory systems to major organs including kidneys, liver, heart, CNS, and lymphatic systems.
• Leads to fatality in pneumonia cases.

Legionnaires' Disease (Cont'd)

• Mortality rates are typically 15-30%, sometimes approaching 50%.
• Most cases involve serogroup 1 (85%) and occasionally serogroups 4 and 6, plus L. micdadei

Legionella - Disease

• Disease severity ranges from mild flu-like symptoms to severe pneumonia and multi-system involvement
• Specific predispositions such as compromised cellular immunity, or pulmonary dysfunction, tend to lead to more severe diseases.

Pontiac Fever

• Incubation period is typically 2 days.
• Characterized by flu-like symptoms including fever and myalgia, which last for 2-5 days.
• A generally mild form, it usually resolves spontaneously and does not typically result in mortality

Reservoirs of Legionella

• Primarily found in most aquatic environments (lakes, rivers, springs, man-made water treatment systems).
• Can survive in chlorinated water.
• Specific reservoirs include the heating and cooling towers of buildings and fountains

Reservoirs of Legionella (Cont'd)

• Colonization is facilitated by their ability to multiply at a wide range of temperatures (20–43°C)
• Ability to survive under specific temperature ranges (40–60°C)
• Adhesion to pipes, plastics, and rubber within the reservoirs
• Ability to live intracellularly in protozoa

Clinical Infections of Legionella

• Intracellular parasites of host cells
• Primarily found in the alveolar macrophages.
• Specific predispositions that increase risk: immunocompromised individuals, patients with chronic lung disease, heavy smokers, and/or alcoholics.

Identifying Legionella

• Respiratory specimens (especially bronchioalveolar lavage, sputum, and bronchial washings) are frequently used
• Urine specimens are tested for antigen, notably successful for serogroup 1 identification, as the antigen persists for long periods.

Identifying Legionella (Cont'd)

• Direct examination can be performed using a special stain to visualize the organism, which is present both intra- and extra-cellularly.
• Direct antigen tests are also available.
• Immunological techniques (indirect fluorescent antibody) used to identify the antigens in specimens.

Culturing Legionella

• Legionella spp. are aerobic bacteria.
• They require special growth media (e.g., BCYE-buffered charcoal yeast extract) enriched with L-cysteine.
• Colonies are characterized as small, pinpoint colonies.
• Acid washing is recommended for reducing contamination with other organisms.
• Sample dilution is often used to quantify the abundance of Legionella.
• Incubation at 35–37°C in normal atmospheric conditions is usually sufficient for at least 7 days.
• Confirmation of growth often requires testing, such as verifying L-cysteine dependence.

Legionella - Growth and Metabolism

• Though flourishing in diverse aquatic environments, Legionella requires iron salts and cysteine (in a buffered medium) in laboratory culture.

Example of DFA Test for Legionella

• A direct fluorescent antibody (DFA) test image provides distinct visualisation of L. pneumophilia in specimens.

Antimicrobial Susceptibility

• Erythromycin and its combination with rifampin are common treatment choices for Legionella infection.
• Numerous alternative treatments are available for varying infections, including doxycycline, trimethoprim–sulfamethoxazole, newer macrolides and or fluoroquinolones.

Bordetella

• Bordet-Gengou bacillus is the common abbreviation for Bordetella, and is known as a small, strict aerobe, gram-negative bacilli
• Pertussis (Latin for severe cough) is another name for whooping cough, a common disease in humans.
• Commonly grown using enriched media with charcoal, starch, blood, and albumin

Bordetella species

• Seven major species: B. pertussis, B. parapertussis, and B. bronchiseptica are amongst the most clinically relevant species.

Bordetella species (cont'd)

• B. pertussis and B. parapertussis are primary causes of whooping cough, mainly in humans.
• B. bronchiseptica an opportunistic pathogen in pneumonia or wound infections, typically in animals (dogs, pigs) but can occur in humans.
• Other minor pathogens include B. avium, B. hinzii, B. holmesii, and B. trematum

Infection of Bordetella pertussis and parapertussis

• Spread through respiratory aerosols.
• The bacteria initially attach and grow on the ciliated respiratory epithelial cells.
• 90% of exposed contacts typically get the disease
• Adults can become transient carriers, either with or without notable symptoms, and the vaccine may be less effective once administered

Bordetella - Virulence factors

• Various virulence factors aiding adhesion and pathogenesis include attachment to ciliated epithelial cells, adenylate cyclase toxin, tracheal cytotoxin, and LPS endotoxin.

Virulence factors

• Filamentous heamagglutinin (FHA)
• Pertussis Toxin (PT) interferes with signal transduction
• Adenylate cyclase toxin increases cAMP levels
• Tracheal cytotoxin causing ciliostasis (ciliary dysfunction)

Clinical Infections and Disease Course

• Whooping cough generally exhibits a 1-2 week incubation period, followed by cold/flu-like symptoms (e.g., sneeze, runny nose).
• Then a catarrhal phase (1-2 weeks, highly contagious) begins, followed by paroxysmal coughing episodes (2-4 weeks).
• The paroxysmal phase is frequently accompanied by a rapid gasp for air, sometimes including vomiting.
• The convalescent phase initiates around 3-4 weeks, characterized by a gradual reduction in coughing episodes.

Whooping Cough (Clinical Syndromes)

• Catarrhal phase: characterised for its first 1-2 weeks; symptoms-like running nose, sneezing, mild fever, and malaise.
• Paroxysmal phase: second phase, lasting for 2-4 weeks, with notable repeated episodes of coughing followed by whooping sound, sometimes vomiting, or difficulty breathing.
• Convalescent phase: third and final phase typically lasting 3-4 weeks and symptoms typically diminish.

Bordetella - Disease

• Whooping cough is contagious and has significant morbidity, and mortality in young children.
• Illnesses are frequently associated with insufficient or inadequate immunization.
• Asymptomatic individuals can serve as reservoirs.

Clinical Infections and Disease Course (cont'd)

• Serious infections in young children can lead to hypoxia or cyanosis due to airway obstruction.
• Convalescent phase is marked with reduced coughing, but complete resolution can take weeks or months.

Diagnosis and Isolation

• Nasopharyngeal samples are collected using specific tools like Calcium alginate, or Dacron swabs.
• These samples are inoculated onto transport media (e.g., Regan-Lowe transport medium), then plated directly (e.g., charcoal agar), and incubated in aerobic conditions at 35°C for 7 days, to rule out contamination by other microorganisms such as Haemophilus .
• Bordet-Gengou agar media with glycerol and sheep blood can also be used for isolation

Colony Morphology

• Colonies of Bordetella are characterized as gram-negative rods or coccobacilli, appearing smooth, with silver-like pinpoint colonies resembling mercury droplets
• Direct fluorescent antibody tests and agglutination tests are often used to confirm the diagnosis of Bordetella.

Differential Characteristics of Bordetella species

• Differential growth results of Bordetella spp. (pertussis, parapertussis, bronchiseptica) on various types of agar to confirm presence of a given species.

Antimicrobial Susceptibility

• Erythromycin is the most common antibiotic for treating Bordetella infections.
• Other alternatives include azithromycin

Test your knowledge on brucellosis in humans and its causative agent, Brucella species, as well as key facts about Haemophilus influenzae. This quiz covers symptoms, treatments, laboratory methods, and unique characteristics of these pathogens. Challenge yourself with these important microbial biology topics!