Content:
◉ Introduction
The members of the genus Haemophilus are small Gram-negative bacilli, which are strict pathogens. The genus derives its name from its essential growth requirement of certain factors present in the blood (Haemophilus: Haem: blood; philos: loving).
The first member of this genus was isolated by Koch in 1883 from a case with conjunctivitis in Egypt and is known as H. aegyptius. The type species of Haemophilus, i.e., H. influenzae, was isolated in 1892 by Pfeiffer from a patient with influenza and was erroneously considered the causative agent of this disease.
Many other Gram-negative bacteria have been included in this genus because of their requirement of one or both of the growth factors present in blood, essential for the growth of the organism. These factors are designated as X (hemin or other porphyrins) and V (nicotinamide adenine dinucleotide (NAD) or NADP).
◉ Classification-Nomenclature
Haemophilus influenzae is a species classified in the genus Haemophilus (contains 16 species) within the family Pasteurellaceae
- Family: Pasteurellaceae
- Genus: Haemophilus.
- Species: H. influenzae (the type species)
◉ Epidemiology
Haemophilus spp. normally inhabit the upper respiratory tract of humans. Asymptomatic colonization with H. influenzae type b is rare. Among H. influenzae strains, there are two broad categories: typeable and nontypeable (NTHi).
Strains are typed based on capsular characteristics. The capsule is composed of a sugar-alcohol phosphate (i.e., polyribitol phosphate) complex. Differences in this complex are the basis for separating encapsulated strains into one of six groups: type a, b, c, d, e, or f. H. influenzae type b (Hib) is most commonly encountered in serious infections in humans.
Non-typeable strains do not produce a capsule and are most commonly encountered as normal inhabitants of the upper respiratory tract.
Although person-to-person transmission plays a key role in infections caused by Haemophilus influenzae, infections caused by other strains and species of Haemophilus likely arise endogenously when a person's own flora gains access to a site normally sterile.
The colonizing organism invades the mucosa and enters the patient’s bloodstream. Encapsulated strains are protected from clearance from host phagocytes. Once in the circulation, the organism is able to spread to additional sites and tissues including the lungs, pericardium, pleura, and meninges.
◉ Pathogenesis
H. influenzae produces no exotoxin. The nonencapsulated organism is a regular member of the normal respiratory microbiota of humans. The capsule is antiphagocytic in the absence of specific anticapsular antibodies.
The polyribose phosphate capsule of type b H. influenzae is the major virulence factor. The carrier rate in the upper respiratory tract for H. influenzae type b was 2-4% in the prevaccine era and is now less than 1%. The carrier rate for nontypeable H. influenzae is 50-80% or higher. Type b H. influenzae causes meningitis, pneumonia and empyema, epiglottitis, cellulitis, septic arthritis, and occasionally other forms of invasive infection.
Although type b can cause chronic bronchitis, otitis media, sinusitis, and conjunctivitis, it does so much less commonly than nontypeable H. influenzae. The blood of many persons older than age 3-5 years is bactericidal for H. influenzae, and clinical infections are less frequent in such individuals. However, bactericidal antibodies have been absent from 25% of adults in the United States, and clinical infections have occurred in adults.
◉ Diseases
H. influenzae used to be the leading cause of meningitis in young children, but the use of the highly effective “conjugate” vaccine has greatly reduced the incidence of meningitis caused by this organism.
It is still an important cause of upper respiratory tract infections (otitis media, sinusitis, conjunctivitis, and epiglottitis) and sepsis in children. It also causes pneumonia in adults, particularly in those with chronic obstructive lung disease.
◉ Morphology and Identification
◉ A. Typical Organisms
In specimens from acute infections, the organisms are short (1.5 μm) coccoid bacilli, sometimes occurring in pairs or short chains.
In cultures, the morphology depends both on the length of incubation and on the medium. At 6-8 hours in rich medium, the small coccobacillary forms predominate. Later there are longer rods, lysed bacteria, and very pleomorphic forms.
Organisms in young cultures (6-18 hours) on enriched medium have a definite capsule. The capsule is the antigen used for “typing” H. influenzae.
◉ B. Culture
On chocolate agar, flat, grayish brown colonies with diameters of 1-2 mm are present aft er 24 hours of incubation. IsoVitaleX in media enhances growth. H. influenzae does not grow on sheep blood agar except around colonies of staphylococci (“satellite phenomenon”).
◉ C. Growth Characteristics
Identification of organisms of the Haemophilus group depends partly on demonstrating the need for certain growth factors called X and V. Factor X acts physiologically as hemin; factor V can be replaced by nicotinamide adenine nucleotide (NAD) or other coenzymes. Colonies of staphylococci on sheep blood agar cause the release of NAD, yielding the satellite growth phenomenon. Carbohydrates are fermented poorly and irregularly.
In addition to serotyping on the basis of capsular polysaccharides, H. influenzae and H parainfluenzae can be biotyped on the basis of the production of indole, ornithine decarboxylase and urease. Most of the invasive infections caused by H. influenzae belong to biotypes I and II (there are a total of eight).
◉ Diagnostic Laboratory Tests
◉ A. Specimens
Specimens consist of expectorated sputum and other types of respiratory specimens, pus, blood, and spinal fluid for smears and cultures depending on the source of the infection.
◉ B. Direct Identification
Commercial kits are available for immunologic detection of H. influenzae antigens in spinal fluid. A positive test result indicates that the fluid contains high concentrations of specific polysaccharide from H. influenzae type b. These antigen detection tests generally are not more sensitive than a Gram stain and therefore are not widely used, especially because the incidence of H. influenzae meningitis is so low.
◉ C. Culture
Specimens are grown on IsoVitaleX-enriched chocolate agar until typical colonies appear. H. influenzae is differentiated from related gram-negative bacilli by its requirements for X and V factors and by its lack of hemolysis on blood agar.
Tests for X (heme) and V (nicotinamide-adenine dinucleotide) factor requirements can be done in several ways. The Haemophilus species that require V factor grow around paper strips or disks containing V factor placed on the surface of agar that has been autoclaved before the blood was added (V factor is heat labile).
Alternatively, a strip containing X factor can be placed in parallel with one containing V factor on agar deficient in these nutrients. Growth of Haemophilus in the area between the strips indicates requirement for both factors. A better test for X factor requirement is based on the inability of H. influenzae (and a few other Haemophilus species) to synthesize heme from δ-aminolevulinic acid.
The inoculum is incubated with the δ-aminolevulinic acid. Haemophilus organisms that do not require X factor synthesize porphobilinogen, porphyrins, protoporphyrin IX, and heme. The presence of red fluorescence under ultraviolet light (~360 nm) indicates the presence of porphyrins and a positive test result.
◉ Antimicrobial Susceptibility Testing And Therapy
◉ 1- Intrinsic Resistance
Haemophilus influenzae shows intrinsic resistance to certain antibiotics, such as macrolides with a 16-atom ring, lincosamides, bacitracin, mecillinam, oxacillin, and glycopeptides. It exhibits moderate sensitivity to macrolides with 14 and 15-atom rings (e.g., erythromycin), streptogramins, and first-generation cephalosporins (e.g., cefalotine).
◉ 1- Acquired Resistance
Resistance to ß-lactams often results from the production of beta-lactamase (TEM type), but it can also arise from mutations in penicillin-binding proteins (PBPs), reducing antibiotic effectiveness. The use of beta-lactamase inhibitors can restore their activity.
Acquired resistance may also affect tetracyclines, phenicols, fluoroquinolones, and trimethoprim-sulfamethoxazole, though the incidence is typically very low.
Note: Care should be taken when preparing inoculum concentrations (0.5 McFarland) for Haemophilus spp.; in particular, betalactamase- producing strains of H. influenzae, as higher suspensions may lead to false-resistant results.