UNIVERSITY OF SANTO TOMAS

Faculty of Pharmacy: 3-G Medical TechnologyGROUP 2

GENUS HAEMOPHILUSSubmitted in partial fulfillment of the requirements for

Microbiology 231

Submitted by: Carlos, Maria Bianca C.

Castillo, John Cyril D.

Castro, Justin Gerard C.

Chua, Albert Y.

Cielo, Angela V.

Collarin, Hannah Camille M.

De Guzman, Masao E.

Genus Haemophilus

General Characteristics

Genus Haemophilus belong to the family Pasteurellaceae. They are characteristically Gram-negative, pleomorphic, coccoid to rod-shaped cells that are nonmotile and facultatively anaerobic. They form nitrites from nitrates (nitrate reducers), and are oxidase and catalase positive. Haemophilus belongs to the HACEK group, along with Aggregatibacter, Cardiobacterium, Eikenella, and Kingella, which resides in the human oral cavity; some species have an enhanced capacity to cause endocarditis.

Colony characteristics

Haemophilus grow in small, round, convex colonies, which may be iridescent, develop in 24 hours on chocolate blood agar. Encapsulated strains appear small, semi-opaque, gray-white, and mucoid on chocolate agar.

Microscopic appearance

Haemophilus is a genus of Gram-negative, pleomorphic, coccobacilli bacteria belonging to the Pasteurellaceae family. While Haemophilus bacteria are typically small coccobacilli, they are categorized as pleomorphic bacteria because of the wide range of shapes they occasionally assume. The genus includes commensal organisms along with some significant pathogenic species such as H. influenzae—a cause of sepsis and bacterial meningitis in young children. The encapsulated strains are subclassified in serovars a-f based on the fine structure of their capsule polysaccharides. Serovar b (Hib) causes most Haemophilus influenzae infections in humans. H. ducreyi is the causative agent of chancroid. All members are either aerobic or facultative anaerobic.

Virulence factors

Capsule

The capsule is the most significant virulence factor present in Haemophilus organisms. H. influenzae, though not all, can express one of six antigenically distinct polysacchaide capsules, designated a through f. The most invasive infections were caused by encapsulated strains of Haemophilus influenza belonging to serotype b (Hib) and occurred primarily in systemic infections in children, whereas most respiratory tract isolates are uncapsulated, referred to as non-typeable. In unvaccinated children, type b is the leading cause of meningitis. The serotype b capsule is a unique polymer composed of ribose, ribitol, and phosphate (polyribitol phosphate [PRP]). The antiphagocytic property and anticomplementary activity of the type b are important factors in virulence.

IgA proteases

Immunoglobulin A proteases interferes with the barrier functions of mucosal IgA antibodies by cleaving at peptide bonds within the hinge region. Secretory IgA is present on human mucosal surfaces of the respiratory tract, areas where H. influenzae has a predilection. H. influenzae is the only member of the genus that produces IgA proteases.

Adhesion factors

Most nonencapsulated strains are adherent to human epithelial cells, while most serotype b strains are not. The lack of this adherent capability of type b strains may explain the tendency of type b strains to cause systemic infections. One example of adhesion protein is the haemagglutinating pili, also called fimbriae, which promote adherence to respiratory mucus and human oropharyngeal epithelial cells, as well as promote interaction with heparin-binding extracellular matrix proteins. Others are Hap (promote adherence and invasion), Hia/Hsf (high-affinity adhesive activity and mediates interaction with a broad array of respiratory epithelial cell types), HMW1/HMW2 (mediate attachment to human epithelial cells found in non-typeable strains), and OapA (required for efficient colonization of the nasopharynx and may play a minor role in adherence).

LPS and OM protein

The LPS comprises two distinct, covalently linked regions, the lipid A and the core oligosaccharide. LPS is an important virulence determinant of H. influenzae, playing a key role in the processes of bacterial colonization, persistence and survival in the human host, but also represents an ideal target for bactericidal as well as endotoxin-neutralizing antibodies. The lipid A portion of H. influenzae LPS is embedded in the outer membrane and mediates the endotoxic effects of the LPS molecule responsible for some of the pathophysiology associated with severe infection. LOS phosphorylcholine (ChoP) may influence invasion via interaction with PAF receptor and stimulates inflammatory response. P2 protein is the most abundant major outer membrane protein of non-typeable H. influenza. It allows the organism to evade clearance by potentially protective antibodies and contributes to the development of chronic infection. P5 protein interacts with a glycoprotein expressed by respiratory epithelial cell.

LPS Phase Variation

LPS phase variation is characterized by the spontaneous loss and gain of oligosaccharide structures present in the outer core. The phase variable expression of LPS biosynthesis genes promotes evasion of antigen-specific host immune defences and allows colonization of different host microenvironments. LPS has been shown to have a paralyzing effect on the sweeping motion of ciliated respiratory epithelium.

Cultivation

Haemophilus species are aerobic or facultative anaerobes. They catabolize D-glucose and other carbohydrates, producing acids. They are non-motile and nonspore-forming. In addition, they are generally oxidase positive except H. ducreyi and reduce nitrates to nitrites. For most species, growth is optimal at a temperature of 33–37 °C in a humid environment. Chocolate agar is the medium of choice for isolating Haemophilus species. They grow best at 35 °C in a 3-5% CO2 atmosphere. Although exogenous X factor is available in SBA, this medium will not support the growth of most Haemophilus species because the V factor is inaccessible inside the RBC’s. However if S. aureus (which secretes V factor) is present on the BAP, Haemophilus species will grow as tiny colonies around the S. aureus colonies. This is called the satellite phenomenon.

Haemophilus isolation agar with bacitracin is a primary plating medium used for the selective isolation of Haemophilus species. Haemophilus Isolation Agar is formulated with Fildes Enrichment and BBL IsoVitaleX enrichment to supply the essential X and/or V growth factors. Horse blood provides appropriate hemolytic reactions to facilitate the differentiation of

Haemophilus species. The antimicrobial agent bacitracin is incorporated to inhibit the growth of bacteria that could mask the presence of Haemophilus species. Haemophilus isolation agar with bacitracin consists of Brain Heart Infusion Agar supplemented with Fildes enrichment, IsoVitaleX enrichment and horse blood. Fildes enrichment is a peptic digest of sheep blood that supplies both X and V factors. IsoVitaleX enrichment is a chemically-defined supplement that provides V factor and other nutrients, such as thiamine and cysteine, to stimulate the growth of Haemophilus species.

Haemophilus Test Medium Agar (HTM Agar) is intended for use in the antimicrobial disc diffusion susceptibility procedure for Haemophilus spp. This medium is Mueller Hinton agar or broth supplemented with X factor (hemin or hematin), V factor (nicotinamide adenine dinucleotide, NAD) and yeast extract. A major advantage of HTM Agar compared with Mueller Hinton Chocolate Agar is optical clarity, permitting zone diameter measurements from the bottom of the dish as is the standard test procedure for nonfastidious organisms on Mueller Hinton Agar. Furthermore, HTM Agar contains low levels of thymidine and is, therefore, suitable for testing sulfamethoxazole/trimethoprim (SXT).

Biochemical Identification of the Species

X & V test

Members of the Haemophilus genus are typically cultured on blood agar plates as all species require at least one of the following blood factors for growth: hemin (X factor) and/or nicotinamide adenine dinucleotide (V factor). Chocolate agar is an excellent Haemophilus growth medium as it allows for increased accessibility to these factors.

X & V Strips

Tests for X factor (heme) and V factor (nicotinamide-adenine dinucleotide) 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.

Satellite Test

Haemophilus is sometimes cultured using the "Staph streak" technique: both Staphylococcus and Haemophilus organisms are cultured together on a single blood agar plate. In this case, Haemophilus colonies will frequently grow in small "satellite" colonies around the larger Staphylococcus colonies because the metabolism of Staphylococcus produces the necessary V factor (NAD) by-products required for Haemophilus growth.

ALA-Porphyrin test

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 organism is inoculated into 0.5 ml of 0.1 M sodium phosphate buffer, pH 6.9, containing 0.08 mM magnesium sulfate and 2 mM δ-aminolevulinic acid and then incubated at 37°C for 4 hours. .

Factor X-independent bacteria incorporate the aminolevulinic acid into the heme biosynthesis pathway and excrete pathway intermediates, such as porphobilinogen and porphyrins. Porphobilinogen is detected by the addition of p-dimethylaminobenzaldehyde (Kovac’s reagent); a red color forms in the lower aqueous phase if porphobilinogen is present. The presence of red fluorescence under a Wood’s light in a dark room (approximately 360 nm) indicates the presence of porphyrins and a positive test; a bluish fluorescence is negative. Haemophilus species that synthesize porphyrins (and thus heme) are not H. influenzae.

Carbohydrate fermentation

Haemophilus species are facultative anaerobes and have both a respiratory and a fermentative type of metabolism. Biochemical test such as carbohydrate fermentation can help furher differentiate the Haemophilus spp. They are capable of fermenting glucose and other carbohydrates, producing acids and sometimes gas.

Table 2. Carbohydrate Fermentation of Haemophilus spp.

Differential Tests for Haemophilus Biogroups

Strains of H. influenza and H. parainfluenzae can be subdivided into biotypes based on biochemical reactions that determine the presence of urease, ornithine decarboxylase, and the production of indole.

Indole test is a biochemical test performed on bacterial species to determine the ability of the organism to split indole from the amino acid tryptophan. Haemophilus influenzae will yield a positive result. Reagents include 0.05 M phosphate buffer (pH 8.0) containing 0.1% tryptophan; after 4 hours incubation at 35 °C, Kovac’s reagent is added and a red color indicates a positive result. Yellow color indicates negative result.

Urease test is a test performed on bacterial species to determine if the bacteria can produce carbon dioxide from urea. A balanced salts solution (0.1% KH2PO4, 0.1% K2HPO4, 0.5% NaCl, and 0.5 ml of a 2% solution phenol red), pH 7.0, is prepared and autoclaved. Development of red color indicates positive result. Christensen’s urea agar slants may also be used.

Ornithine decarboxylase test is used to determine the ability of the bacteria to catalyze the decarboxylation of ornithine. Standard Moeller’s ornithine decarboxylase broth is used. Heavy inoculum is used and each tube is overlaid with sterile mineral oil.

Pathogenesis and Clinical Manifestations

Haemophilus influenzae

Invasive infection – caused by encapsulated strains of H. influenzae

Meningitis – inflammation of the protective membranes covering the brain and spinal cord; treatment with third generation cefalosporin such as cefotaxime or ceftriaxone, vancomycin, ampicillin

Epiglottitis – inflammation of the epiglottis, causes the epiglottis to either obstruct or completely close off the windpipe thus resulting to interference when breathing; treatment with antibiotics such as second or third generation cephalosporins (either alone or in combination with penicillin or ampicillin for streptococcal coverage

Tracheitis – inflammation of the trachea, may cause obstruction of air passage; treatment with third-generation cephalosporin (eg. cefotaxime or ceftriaxone) and a penicillinase-resistant penicillin

Cellulitis – a diffuse inflammation of connective tissue with severe inflammation of dermal and subcutaneous layers of the skin; treatment with oral phenoxymethylpenicillin or intravenousbenzylpenicillin, or ampicillin/amoxicillin

Localized infection – caused by nonencapsulated strains of H. influenzae

Sinusitis – inflammation of paranasal sinuses; treatment with amoxicillin, fluoroquinolones, macrolide antibiotics such as clarithromycin and doxycycline, are used in patients who are allergic to penicillins, surgery, corticosteroids

Otitis media – inflammation of the middle ear, located between the tympanic membrane and the inner ear, including a duct known as the Eustachian tube; treatment with five day treatment with amoxicillin or amoxicillin-clavulanate

Haemophilus aegyptius

Conjunctivitis – acute inflammation of the conjunctiva; treated with antibiotics, anti histamines for allergy, eye drops

Brazilian purpuric fever (BPF) – preceded by conjunctivitis, prostrating high fevers and chills, vomiting and gastrointestinal complaints progress rapidly to extensive purpura (red or purple discolorations of the skin), septic shock and acidosis; treated with ampicillin plus chloramphenicol but the mortality rate is around 70% even with therapy

Haemophilus ducreyi

Chancroid is a highly communicable sexually transmitted genital ulcer disease (GUD). It is commonly referred to as soft chancre, in contrast to the hard chancre of syphilis. After 4 to 14 days of incubation period, painful lesions with irregular edges appear on the genitalia or perianal areas. Treatments include single oral dose (1 gram) of azithromycin, a single IM dose of ceftriaxone, oral erythromycin for seven days.

Other species

Haemophilus parainfluenzae may occasionally cause pneumonia, inflammatory condition of the lung specifically in the alveoli and filling of alveoli with fluid, and endocarditis, where the mitral valve is the primary site of infection. H. parahaemolyticus may be a cause of some cases of pharyngitis.

References

From books:

Adelberg, Jawetz, & Melnick. (2007). Medical Microbiology, 24th Edition. The McGraw-Hill

Companies: USA.

Dworkin, M. (ed.) The Prokaryotes: A handbook on the biology of bacteria, 3d ed., Vol. 6:

Proteobacteria: Gamma subclass. Springer Science, NY: USA

Gillespie, S. H. & Hawkey, P. M. (ed.). (2006). Principles and practice of clinical bacteriology.

2nd Edition. John Wiley & Sons Ltd: United Kingdom.

Kayser, F. H. (2005). Medical Microbiology. Thieme: Stuttgart, Germany.

From the internet:

State Key Laboratory for Moleclular Virology and Genetic Engineering. (2003). Virulence factors

of pathogenic bacteria. Retrieved on September 11, 2010, from, http://www.mgc.ac.cn/cgi

bin/VFs/vfs.cgi?VFID=VF0040#VF0040

St. Louis Community College website. (2008). Biochemical tests. Retrieved on September 11,

2010, from, http://users.stlcc.edu/kkiser/biochem.html