The Haemophilus Species



By:Dr. Kareema Amine Al-Khafajii ,Assistant Professor, Babylon University ,College Of Medicine, Department Of Microbiology .

The Haemophilus Species

This is a group of small, gram-negative, pleomorphic bacteria that require enriched media, usually containing blood or its derivatives, for isolation. Haemophilus influenzae type b is an important human pathogen; Haemophilus ducreyi, a sexually transmitted pathogen, causes chancroid; other Haemophilus species are among the normal flora of mucous membranes and only occasionally cause disease.

Classification and Characteristics

 Bacteria of the genus Haemophilus are small, non-motile gram negative bacilli and coccobacilli, and are fastidious by nature.  They characteristically require certain growth factors, namely heme (X factor) and nicotine adenine dinucleotide (NAD or V factor). These growth requirements are used in the clinical setting to identify suspect organisms to the species level. Haemophilus spp. grow best at a temperature between 35 °C and 37°C, at a slightly alkaline pH of 7.6.  Laboratory growth of H. influenzae is typically achieved in 5% CO2 atmospheric conditions on chocolate blood agar, which provides the previously mentioned X and V factors.   

Haemophilus influenzae

Haemophilus species are typically considered to be normal flora in the upper respiratory tract of humans. They are found in the nasopharynx of approximately 75% of healthy individuals.  Normal flora H .influenzae are non-encapsulated. Less than 5% of healthy individuals harbour an encapsulated strain of Haemophilus influenzae, which serotypes as type b that considered to be the most common cause of bacterial meningitis in young children ages 3 months to 6 years. a polysaccharide capsule which prevents phagocytosis by the body’s immune cells. Non-encapsulated strains may possess a variety of cell surface proteins, fimbriae and pili which may aid in host cell attachment.

Transmission and Infection

Transmission of H. influenzae is primarily through person to person contact via respiratory droplets contaminated with the bacteria. A person's own endogenous strains could colonize another area of the body, and become pathogenic in that area. H. influenzae could also become an opportunistic pathogen in the upper respiratory tract in an immunocompromised host. The use of the H. influenzae type b vaccine in children has virtually eradicated meningitis caused by the bacteria in many countries, but infections may still be seen in infants too young to be vaccinated or in other unvaccinated children.

Clinical Manifestations

A wide variety of infections and clinical manifestations may be seen with H. influenzae, and their severity varies depending upon whether the bacteria is of the encapsulated or non-encapsulated variety.  Clinical manifestation is also dependent on factors such as location of infection, and the immune status of the host.  

Non-encapsulated strains may cause the following:    

• sinusitis    

• pneumonia    

• acute axacerbation of COPD

• otitis media

Encapsulated strains may cause the following:     

• meningitis     

• septic arthritis     

• pneumonia    

• conjunctivitis     

• cellulitis

• epiglottitis

Diagnosis

*Specimens should be cultured to chocolate agar plates and incubated in 5-10% CO2 at 35-37 °C for 24- 72 hours where they will produce small or medium grey, mucoid (if encapsulated) colonies.  A characteristic "mousy" odor is often detected.  *Investigations may begin with the appearance of pale Gram negative coccobacilli or bacilli on a direct Gram stain. It should be noted that H. parainfluenzae may be pleomorphic with long filaments but it may also resemble H. influenzae and requires only NAD (V) supplementation. 

*One identification scheme involves plating the specimen to a blood agar plate and adding a streak line of Staph. aureus the plate is then incubated at 5% CO2, 35 °C, for 16-18 hours.  S. aureus creates NAD (V factor) as a metabolic byproduct.  Since blood agar plates contain hemin (X factor), a presumptive identification can be made when bacterial colonies are seen satelliting around the S. aureus.

Treatment

In the past, ampicillin was an effective treatment for H. influenzae infection, but many strains are now resistant to beta lactam antibiotics. For type b encapsulated strains, cefuroxime is recommended for treatment as it has the ability to cross the BBB.  For non-encapsulated strains causing upper respiratory infections, amoxicillin coupled with a beta lactamase inhibitor (e.g. clavulinic acid) is often employed.

Haemophilus ducreyi

H. ducreyi causes chancroid (soft chancre), a sexually transmitted disease. Chancroid consists of a ragged ulcer on the genitalia, with marked swelling and tenderness. The regional lymph nodes are enlarged and painful. The disease must be differentiated from syphilis, herpes simplex infection, and lymphogranuloma venereum.

H ducreyi requires X factor but not V factor. There is no permanent immunity following chancroid infection. Treatment with intramuscular ceftriaxone, oral trimethoprim-sulfamethoxazole, or oral erythromycin often results in healing in 2 weeks.

The Bordetellae

There are several species of bordetella. B. pertussis, a highly communicable and important pathogen of humans, causes whooping cough (pertussis). B. parapertussis can cause a similar disease. B. bronchiseptica causes diseases in animals. The organisms are minute gram-negative coccobacilli resembling H influenzae. With toluidine blue stain, bipolar metachromatic granules can be demonstrated. A capsule is present.

Culture

Primary isolation of B. pertussis requires enriched media. Bordet-Gengou medium (potato-blood-glycerol agar) that contains penicillin G to inhibit other respiratory flora but permit growth of B pertussis, however, a charcoal-containing medium similar to that used for L. pneumophila is preferable. The plates are incubated at 35–37 °C for 3–7 days in a moist environment. The small, faintly staining gram-negative rods are identified by immunofluorescence staining.

The organism is nonmotile, a strict aerobe and forms acid but not gas from glucose and lactose. It does not require X and V factors on subculture. Hemolysis of blood-containing medium is associated with virulent B. pertussis.

Pathogenesis:

B. pertussis produces a number of factors that are involved in the pathogenesis of disease. The filamentous hemagglutinin mediates adhesion to ciliated epithelial cells. Pertussis toxin promotes lymphocytosis, sensitization to histamine, and has ADP-ribosylating activity, with an A/B structure and mechanism of action similar to that of cholera toxin. The filamentous hemagglutinin and pertussis toxin are secreted proteins and are found outside of the B. pertussis cells. The tracheal cytotoxin inhibits DNA synthesis in ciliated cells. Pili probably play a role in adherence of the bacteria to the ciliated epithelial cells. The lipopolysaccharide in the cell wall may also be important in causing damage to the epithelial cells of the respiratory tract.

Transmission is largely by the respiratory route from early cases and possibly via carriers. The organism adheres to and multiplies rapidly on the epithelial surface of the trachea and bronchi and interferes with ciliary action.

Clinical Findings

After an incubation period of about 2 weeks, the "catarrhal stage" develops, with mild coughing and sneezing. During this stage, large numbers of organisms are sprayed in droplets, and the patient is highly infectious but not very ill. During the "paroxysmal" stage, the cough develops its explosive character and the characteristic "whoop" upon inhalation. This leads to rapid exhaustion and may be associated with vomiting, cyanosis, and convulsions. The "whoop" and major complications occur predominantly in infants; paroxysmal coughing predominates in older children and adults. The white blood count is high (16,000–30,000/[pic]L), with an absolute lymphocytosis. Convalescence is slow. B pertussis is a common cause of prolonged (4–6 weeks) cough in adults. Rarely, whooping cough is followed by the serious and potentially fatal complication of encephalitis.

Diagnostic Laboratory Tests

Specimens

A saline nasal wash is the preferred specimen. Nasopharyngeal swabs or cough droplets expelled onto a "cough plate" held in front of the patient's mouth during a paroxysm are sometimes used but are not as good as the saline nasal wash. The direct fluorescent antibody test can be used to examine nasopharyngeal swab specimens. PCR is the most sensitive method to diagnosis pertussis. Serologic tests on patients are of little diagnostic help because a rise in agglutinating or precipitating antibodies does not occur until the third week of illness.

Immunity

Recovery from whooping cough or immunization is followed by immunity. Second infections may occur but are mild; reinfections occurring years later in adults may be severe. It is probable that the first defense against B. pertussis infection is the antibody that prevents attachment of the bacteria to the cilia of the respiratory epithelium.

Treatment

B pertussis is susceptible to several antimicrobial drugs in vitro. Administration of erythromycin during the catarrhal stage of disease promotes elimination of the organisms and may have prophylactic value. Treatment after onset of the paroxysmal phase rarely alters the clinical course. Oxygen inhalation and sedation may prevent anoxic damage to the brain.

Prevention

Every infant should receive three injections of pertussis vaccine during the first year of life followed by a booster series for a total of five doses. There are multiple acellular pertussis vaccines. The acellular vaccines have one to five antigens. Vaccines with three to five antigens yield a better immune response than those with one or two antigens. Pertussis vaccine is usually administered in combination with toxoids of diphtheria and tetanus (DTP). Prophylactic administration of erythromycin for 5 days may also benefit unimmunized infants or heavily exposed adults.

B. parapertussis: This organism may produce a disease similar to whooping cough, but it is generally less severe. The infection is often subclinical. B. parapertussis grows more rapidly than typical B pertussis and produces larger colonies. It also grows on blood agar. B parapertussis has a silent copy of the pertussis toxin gene.

B. bronchiseptica: inhabits the respiratory tracts of canines and cause pneumonitis. It causes snuffles in rabbits and atrophic rhinitis in swine. It is infrequently responsible for chronic respiratory tract infections in humans, primarily in individuals with underlying diseases. It grows on blood agar medium.

Legionellae: the motile, aerobic, fastidious rod-shaped Gram-negative bacterium that thrives in central heating and air conditioning systems and can cause Legionnaires' disease.

Culture: Legionellae can be grown on complex media such as buffered charcoal-yeast extract (BCYE) agar with [pic]-ketoglutarate, at pH 6.9, temperature 35 °C. Antibiotics can be added to make the medium selective for legionella. It grow slowly; visible colonies are usually present after 3 days of incubation. Colonies that appear after overnight incubation are not legionella. Colonies are round or flat with entire edges. They vary in color from colorless to iridescent pink or blue and are translucent or speckled. Variation in colony morphology is common, and the colonies may rapidly lose their color and speckles. Many other genera of bacteria grow on BCYE medium and must be differentiated from legionella by Gram staining and other tests.

Legionellae in blood cultures usually require 2 weeks or more to grow. Colonies can be seen on the agar surface of the biphasic medium.

Growth Characteristics

The legionellae are catalase-positive. L pneumophila is oxidase-positive; the other legionellae are variable in oxidase activity. Most legionellae produce gelatinase and [pic]-lactamase.

Antigens & Cell Products

Antigenic specificity of L pneumophila is thought to be due to complex antigenic structures. There are more than ten serogroups of L pneumophila; serogroup 1 was the cause of the 1976 outbreak of Legionnaires' disease and remains the most common serogroup isolated from humans. Legionella species cannot be identified by serogrouping alone, because there is cross-reactive antigenicity among different species. The legionellae make proteases, phosphatase, lipase, DNase, and Rnase.

Pathology & Pathogenesis

Legionellae are ubiquitous in warm moist environments. They are found in lakes, streams, and other bodies of water. Infection of debilitated or immunocompromised humans commonly follows inhalation of the bacteria from aerosols generated from contaminated air-conditioning systems, shower heads, and similar sources. L pneumophila usually produces a lobar, segmental, or patchy pulmonary infiltration. Histologically, the appearance is similar to that produced by many other bacterial pathogens. Acute purulent pneumonia involving the alveoli is present with a dense intra-alveolar exudate of macrophages, polymorphonuclear leukocytes, red blood cells, and proteinaceous material. Most of the legionellae in the lesions are within phagocytic cells. There is little interstitial infiltration and little or no inflammation of the bronchioles and upper airways.

L pneumophila readily enters and grows within human alveolar macrophages and monocytes and is not effectively killed by polymorphonuclear leukocytes. Once inside the cell, the individual bacteria are within phagosomal vacuoles, but the defense mechanisms of the macrophage cells stop at that point. Instead, the phagosomal vacuoles fail to fuse with lysosomal granules. The phagocyte oxidative metabolic burst is reduced. The bacteria multiply within the vacuoles until they are numerous, the cells are destroyed, the bacteria are released, and infection of other macrophages then occurs.

Clinical Findings

Asymptomatic infection is common in all age groups, as shown by elevated titers of specific antibodies. The incidence of clinically significant disease is highest in men over age 55 years. Factors associated with high risk include smoking, chronic bronchitis and emphysema, steroid and other immunosuppressive treatment (as in renal transplantation), cancer chemotherapy, and diabetes mellitus. When pneumonia occurs in patients with these risk factors, legionella should be investigated as the cause.

Infection may result in febrile illness of short duration or in a severe, rapidly progressive illness with high fever, chills, malaise, nonproductive cough, hypoxia, diarrhea, and delirium. Chest x-rays reveal patchy, often multilobar consolidation. There may be leukocytosis, hyponatremia, hematuria (and even renal failure), or abnormal liver function. The diagnosis is based on the clinical picture and exclusion of other causes of pneumonia by laboratory tests. Demonstration of legionella in clinical specimens can rapidly yield a specific diagnosis.

L pneumophila also produces a disease called "Pontiac fever,". The syndrome is characterized by fever and chills, myalgia, malaise, and headache that develop over 6–12 hours. Dizziness, photophobia, neck stiffness, and confusion also occur. Respiratory symptoms are much less prominent in Pontiac fever than in Legionnaire's disease and include mild cough and sore throat.

Diagnostic Laboratory Tests

Specimens

In human infections, the organisms can be recovered from bronchial washings, pleural fluid, lung biopsy specimens, or blood. Isolation of legionella from sputum is more difficult because of the predominance of bacteria of the normal flora. Legionella is rarely recovered from other anatomic sites. Antigens can be demonstrated in the patient's urine by immunologic methods. Legionellae are not demonstrable in Gram-stained smears of clinical specimens. Direct fluorescent antibody tests of specimens can be diagnostic, but the test has low sensitivity compared with culture on BCYE agar.

Immunity

Infected patients make antibodies against legionella, but the peak antibody response may not occur until 4–8 weeks after infection. The cell-mediated response is important in protective immunity because of the intracellular infection and growth of legionella.

Treatment

Legionellae are susceptible to erythromycin which is effective even in immunocompromised patients. Rifampin, been used in patients whose response to treatment was delayed. Assisted ventilation may be necessary, and management of shock is essential.

Epidemiology & Control

The natural habitats for legionellae are lakes, streams, rivers, and especially thermally heated bodies of water and soil. Legionellae grow best in warm water in the presence of amebas and water bacteria. They proliferate in amebas much as they do in pulmonary macrophages in the lung.

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