Chapter 18: Haemophilus, Bordetella, Brucella, and Francisella

This is a group of small, gram-negative, pleomorphic bacteria that require enriched media, usually containing blood or its derivatives, for isolation. Haemophilus influenzae is a major human pathogen; Haemophilus ducreyi, a sexually transmitted pathogen, causes chancroid; six other Haemophilus species are among the normal microbiota of mucous membranes and only occasionally cause human disease. Haemophilus aphrophilus and Haemophilus paraphrophilus have been combined into a single species Aggregatibacter aphrophilus; likewise, Haemophilus segnis is now a member of the Aggregatibacter genus (Table 18-1).

H influenzae is found on the mucous membranes of the upper respiratory tract in humans. It is an important cause of meningitis in unvaccinated children and causes upper and lower respiratory tract infections in children and adults.

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 and very pleomorphic forms.

Some organisms in young cultures (6–18 hours) on enriched medium express a definite capsule. The capsule is the antigen used for “typing” H influenzae (see later discussion).

B. Culture

On chocolate agar, flat, grayish, translucent colonies with diameters of 1–2 mm are present after 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”). Haemophilus haemolyticus and Haemophilus parahaemolyticus are hemolytic variants of H influenzae and Haemophilus parainfluenzae, respectively.

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 dinucleotide (NAD) or other coenzymes. Colonies of staphylococci on sheep blood agar cause the release of NAD, yielding the satellite growth phenomenon. The requirements for X and V factors of various Haemophilus species are listed in Table 18-1. Carbohydrate fermentation is useful in species identification as is the presence or absence of hemolysis.

In addition to serotyping on the basis of capsular polysaccharides (see later discussion), 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).

Antigenic Structure

Encapsulated H influenzae contains capsular polysaccharides (molecular weight >150,000) of one of six types (a–f). The capsular antigen of type b is a polyribitol ribose phosphate (PRP). Encapsulated H influenzae can be typed by slide agglutination, coagglutination with staphylococci, or agglutination of latex particles coated with type-specific antibodies. A capsule swelling test with specific antiserum is analogous to the quellung test for pneumococci. Typing can also be done by immunofluorescence. Most H influenzae organisms in the normal microbiota of the upper respiratory tract are not encapsulated and are referred to as nontypeable (NTHi).

The somatic antigens of H influenzae consist of outer membrane proteins. Lipooligosaccharides (endotoxins) share many structures with those of neisseriae.

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 2–5% in the prevaccine era and is now less than 1%. The carrier rate for NTHi 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. NTHi tends to cause chronic bronchitis, otitis media, sinusitis, and conjunctivitis after breakdown of normal host defense mechanisms. The carrier rate for the encapsulated types a and c to f is low (1–2%), and these capsular types rarely cause disease. Although type b can cause chronic bronchitis, otitis media, sinusitis, and conjunctivitis, it does so much less commonly than NTHi. Similarly, NTHi only occasionally causes invasive disease (~5% of cases).

Clinical Findings

H influenzae type b enters by way of the respiratory tract. There may be local extension with involvement of the sinuses or the middle ear. H influenzae, mostly nontypeable, and pneumococci are two of the most common etiologic agents of bacterial otitis media and acute sinusitis. Lower respiratory tract infections such as bronchitis and pneumonia may be seen in patients with conditions that diminish mucociliary clearance. Examples include smoking, chronic obstructive lung disease, and cystic fibrosis. Encapsulated organisms may reach the bloodstream and be carried to the meninges or, less frequently, may establish themselves in the joints to produce septic arthritis. Before the use of the conjugate vaccine, H influenzae type b was the most common cause of bacterial meningitis in children aged 5 months to 5 years in the United States. Clinically, it resembles other forms of childhood meningitis, and diagnosis rests on bacteriologic demonstration of the organism.

Occasionally, a fulminating obstructive laryngotracheitis with swollen, cherry-red epiglottis develops in young children and requires prompt tracheostomy or intubation as a lifesaving procedure. Pneumonitis and epiglottitis caused by H influenzae may follow upper respiratory tract infections in small children and old or debilitated people. Adults may have bronchitis or pneumonia caused by H influenzae.

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. 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. Their use is discouraged in all but limited resource settings where disease prevalence remains high. A Gram stain of H influenzae in sputum is depicted in Figure 18-1. Nucleic acid amplification methods have been developed by some laboratories and may soon be commercially available for direct detection from cerebrospinal fluid and lower respiratory tract infections.

C. Culture

Specimens are grown on IsoVitaleX-enriched chocolate agar until typical colonies appear (see above). 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 (see Table 18-1).

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. Haemophilus species that synthesize porphyrins (and thus heme) are not H influenzae (see Table 18-1).

Immunity

Infants younger than age 3 months may have serum antibodies transmitted from their mothers. During this time, H influenzae infection is rare, but subsequently, the anti­bodies are lost. Children often acquire H influenzae infections, which are usually asymptomatic but may be in the form of respiratory disease or meningitis. H influenzae was the most common cause of bacterial meningitis in children from 5 months to 5 years of age until the early 1990s when the conjugate vaccines became available (see later discussion). By age 3–5 years, many unimmunized children have naturally acquired anti-PRP antibodies that promote complement-dependent bactericidal killing and phagocytosis. Immunization of children with H influenzae type b conjugate vaccine induces the same antibodies.

There is a correlation between the presence of bactericidal antibodies and resistance to major H influenzae type b infections. However, it is not known whether these antibodies alone account for immunity. Pneumonia or arthritis caused by infection with H influenzae can develop in adults with such antibodies.

Treatment

The mortality rate for individuals with untreated H influenzae meningitis may be up to 90%. Many strains of H influenzae type b are susceptible to ampicillin, but up to 25% produce a β-lactamase under control of a transmissible plasmid and are resistant. Essentially all strains are susceptible to the third-generation cephalosporins and carbapenems. Cefotaxime given intravenously gives excellent results. Prompt diagnosis and antimicrobial therapy are essential to minimize late neurologic and intellectual impairment. Prominent among late complications of H influenzae type b meningitis is the development of a localized subdural accumulation of fluid that requires surgical drainage. Up to 27% of NTHi in the United States also produce β-lactamases.

Epidemiology, Prevention, and Control

Encapsulated H influenzae type b is transmitted from person to person by the respiratory route. H influenzae type b disease can be prevented by administration of Haemophilus b conjugate vaccine to children. Currently, there are three PRP polysaccharide-protein monovalent conjugate vaccines (polysaccharide linked to outer membrane protein complex) available for use in the United States: PRP-OMP (PedvaxHIB, Merck and Co., Inc.), PRP-T (ActHIB, Sanofi Pasteur, Inc.), and PRP-T (Hiberix, GlaxoSmithKline). In PRP-OMP, the outer membrane protein complex of Neisseria meningitidis serogroup B is the protein conjugate, whereas for PRP-T it is tetanus toxoid. There are also three combination vaccines that contain H influenzae type b conjugate vaccine. These are: PRP-OMP-HepB (Merck and Co., Inc.), DTaP-IPV/PRP-T (diphtheria, acellular pertussis, and inactivated polio are added to PRP-T; Sanofi Pasteur), and MenCY/PRP-T (meningococcal C and Y vaccine added to PRP-T; GlaxoSmithKline). The reader is referred to the reference by Briere for a complete discussion of these vaccines. Beginning at age 2 months, all children should be immunized with one of the conjugate vaccines. Depending on which vaccine product is chosen, the series consists of three doses at 2, 4, and 6 months of age or two doses given at 2 and 4 months of age. An additional booster dose is given sometime between 12 and 18 months of age. Monovalent conjugate vaccines can be given at the time of other vaccine administration such as DTaP (diphtheria, tetanus, and acellular pertussis). Widespread use of H influenzae type b vaccine has reduced the incidence of H influenzae type b meningitis in children by more than 95%. The vaccine reduces the carrier rates for H influenzae type b.

Contact with patients with H influenzae type b clinical infection poses little risk for adults but presents a definite risk for nonimmune siblings and other nonimmune children younger than age 4 years who are close contacts. Prophylaxis with rifampin is recommended for such children.

This organism was formerly called the Koch-Weeks bacillus and it is associated with highly communicable form of conjunctivitis (pinkeye) in children. Haemophilus aegyptius is closely related to H influenzae biotype III, the causative agent of Brazilian purpuric fever. The latter is a disease of children characterized by fever, purpura, shock, and death. In the past, these infections were mistakenly attributed to H aegyptius.

Organisms belonging to the species H aphrophilus and H paraphrophilus have been combined into the same species, and the name was changed to A aphrophilus. H segnis and Actinobacillus actinomycetemcomitans have also been added to the genus Aggregatibacter. A aphrophilus isolates are often encountered as causes of infective endocarditis and pneumonia. These organisms are present in the oral cavity as part of the normal respiratory microbiota along with other members of the HACEK (Haemophilus species, Actinobacillus/Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group (see Chapter 16).

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.

The small, gram-negative rods occur in strands in the lesions, usually in association with other pyogenic microorganisms. H ducreyi requires X factor but not V factor. It is grown best from scrapings of the ulcer base that are inoculated onto chocolate agar containing 1% IsoVitaleX and vancomycin, 3 μg/mL; the agar is incubated in 10% CO₂ at 33°C. Nucleic acid amplification methods are more sensitive than culture. There is no permanent immunity after chancroid infection. The recommended treatment by the Centers for Disease Control and Prevention is 1 g of azithromycin taken orally. Other treatment regimens include intramuscular ceftriaxone, oral ciprofloxacin, or oral erythromycin; healing results in 2 weeks.

H haemolyticus is the most markedly hemolytic organism of the group in vitro; it occurs both in the normal nasopharynx and in association with rare upper respiratory tract infections of moderate severity in childhood. H parainfluenzae resembles H influenzae and is a normal inhabitant of the human respiratory tract; it has been encountered occasionally in infective endocarditis and in urethritis.

Concept Checks

• Haemophilus species are pleomorphic, gram-negative rods that require either X (hemin) or V (NAD) factors or both for growth. Most of the species in this genus are colonizers of the upper respiratory tract of humans.

• H influenzae is the major pathogen in the group, and strains that are encapsulated, especially serotype b, are more virulent, causing invasive disease, including bacteremia and meningitis in unprotected individuals.

• H influenzae type b, once a significant cause of childhood morbidity and mortality, is now rare in industrialized countries that routinely vaccinate children with one of two available conjugate vaccines.

• H aphrophilus and H paraphrophilus have been combined into a single genus and species, A aphrophilus. Other members of the Aggregatibacter genus include A actinomycetemcomitans and A segnis. These organisms are associated with a variety of infections including endocarditis.

• H ducreyi is associated with the sexually transmitted disease chancroid.

There are several species of Bordetella. Bordetella pertussis, a highly communicable and important pathogen of humans, causes whooping cough (pertussis). Bordetella parapertussis can cause a similar disease. Bordetella bronchiseptica (Bordetella bronchicanis) causes diseases in animals, such as kennel cough in dogs and snuffles in rabbits, and only occasionally causes respiratory disease and bacteremia in humans. Newer species and their disease associations include Bordetella hinzii (bacteremia, respiratory illness, arthritis), Bordetella holmesii (bacteremia among immunosuppressed patients), and Bordetella trematum (wound infections and otitis media). B pertussis, B parapertussis, and B bronchiseptica are closely related, with 72–94% DNA homology and very limited differences in multilocus enzyme analysis; the three species might be considered three subspecies within a species.

Morphology and Identification

A. Typical Organisms

The organisms are minute, gram-negative coccobacilli resembling H influenzae. A capsule is present.

B. Culture

Primary isolation of B pertussis requires enriched media. Bordet-Gengou medium (potato-blood-glycerol agar) that contains penicillin G, 0.5 μg/mL, can be used; however, a charcoal-containing medium supplemented with horse blood, cephalexin, and amphotericin B (Regan-Lowe) is preferable because of the longer shelf life. The plates are incubated at 35–37°C for 3–7 days aerobically in a moist environment (eg, a sealed plastic bag). The small, faintly staining gram-negative rods are identified by immunofluorescence staining. B pertussis is nonmotile.

C. Growth Characteristics

The organism is a strict aerobe and it is oxidase and catalase positive but nitrate, citrate, and urea negative, the results of which are useful for differentiating among the other species of bordetellae. It does not require X and V factors on subculture.

Antigenic Structure, Pathogenesis, and Pathology

B pertussis produces a number of factors that are involved in the pathogenesis of disease. One locus on the B pertussis chromosome acts as a central regulator of virulence genes. This locus has two Bordetella operons, bvgA and bvgS. The products of the A and S loci are similar to those of known two-component regulatory systems. bvgS responds to environmental signals, and bvgA is a transcriptional activator of the virulence genes. Filamentous hemagglutinin, a large surface protein, and fimbriae (surface appendages) mediate adhesion to ciliated epithelial cells and are essential for tracheal colonization. Pertussis toxin (a classic A/B structure toxin) promotes lymphocytosis, sensitization to histamine, and enhanced insulin secretion by means of adenosine diphosphate–ribosylating activity that disrupts function of signal transduction in many cell types. The filamentous hemagglutinin and pertussis toxin are secreted proteins and are found outside of the B pertussis cells. Adenylate cyclase toxin (ACT), dermonecrotic toxin (DNT), and hemolysin also are regulated by the bvg system. ACT is an important virulence factor that inhibits phagocyte function but the role of DNT in pertussis is unknown. The tracheal cytotoxin is not regulated by bvg and kills respiratory epithelial cells in vitro. The lipooligosaccharide in the cell wall may also be important in causing damage to the epithelial cells of the upper respiratory tract.

B pertussis survives for only brief periods outside the human host. There are no vectors. 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. The blood is not invaded. The bacteria liberate the toxins and substances that irritate surface cells, causing coughing and marked lymphocytosis. Later, there may be necrosis of parts of the epithelium and polymorphonuclear infiltration, with peribronchial inflammation and interstitial pneumonia. Secondary invaders such as staphylococci or H influenzae may give rise to bacterial pneumonia. Obstruction of the smaller bronchioles by mucous plugs results in atelectasis and diminished oxygenation of the blood. This probably contributes to the frequency of convulsions in infants with whooping cough.

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/μL), 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 neurological complications (seizures and encephalopathy). Several types of adenovirus and Chlamydia pneumoniae can produce a clinical picture resembling that caused by B pertussis.

Diagnostic Laboratory Tests

A. Specimens

Nasopharyngeal (NP) swabs or NP aspirates using saline are the preferred specimens. Swabs should be either dacron or rayon tipped and not calcium alginate, as it inhibits the polymerase chain reaction (PCR), nor cotton, as cotton kills the organisms. For adults, cough droplets expelled directly onto a “cough plate” held in front of the patient’s mouth during a paroxysm is a less desirable method of specimen collection.

B. Direct Fluorescent Antibody Test

The fluorescent antibody (FA) reagent can be used to examine nasopharyngeal swab specimens. However, false-positive and false-negative results may occur; the sensitivity is about 50%. The FA test is most useful in identifying B pertussis after culture on solid media.

C. Culture

NP aspirates or swabs are cultured on solid media (see earlier discussion). The antibiotics in the media tend to inhibit other respiratory microbiota but permit growth of B pertussis. Organisms are identified by immunofluorescence staining or by slide agglutination with specific antiserum.

D. Polymerase Chain Reaction

PCR and other nucleic acid amplification methods are the most sensitive methods to diagnose pertussis. Primers for both B pertussis and B parapertussis should be included. Where available, a nucleic acid amplification test should replace the direct FA tests. Existing primer targets may cross-react with other Bordetella species.

E. Serology

Production of IgA, IgG, and IgM antibodies occurs after exposure to B pertussis and these antibodies can be detected by enzyme immunoassays. Serologic tests on patients are of little diagnostic help acutely because a rise in agglutinating or precipitating antibodies does not occur until the third week of illness. Serology may be useful in evaluating patients presenting between 2 and 4 weeks of illness. A single serum with high-titer anti-PT IgG may be helpful in diagnosing the cause of a long-term cough, that is, one of greater than 4 weeks’ duration.

Immunity

Recovery from whooping cough or immunization is followed by immunity that is not lifelong. Second infections may occur but are usually milder; 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. Antibodies to PT are highly immunogenic.

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 booster series for a total of five doses. Multiple acellular pertussis vaccines are licensed in the United States and elsewhere. Use of these vaccines is recommended. The acellular vaccines have at least two of the following antigens: inactivated pertussis toxin, filamentous hemagglutinin, fimbrial proteins, and pertactin.

Because different vaccines have different antigens, the same product should be used throughout an immunization series. Pertussis vaccine is usually administered in combination with toxoids of diphtheria and tetanus (DTaP). Five doses of pertussis vaccine are recommended before school entry. The usual schedule is administration of doses at 2, 4, 6, and 15–18 months of age and a booster dose at 4–6 years of age. In 2005, it was recommended by the Advisory Committee on Immunization Practices that all adolescents and adults receive a single booster dose of tetanus, diphtheria, and acellular pertussis (Tdap) to replace the booster dose of tetanus and diphtheria toxoids alone (Td). A strategy to control disease in infants <6 months of age is to vaccinate pregnant women with Tdap. Several acellular pertussis vaccines are available in the United States for use in adolescents and adults.

Prophylactic administration of erythromycin for 5 days may also benefit unimmunized infants or heavily exposed adults.

Epidemiology and Control

Whooping cough is endemic in most densely populated areas worldwide and occurs intermittently in epidemics. The source of infection is usually a patient in the early catarrhal stage of the disease. Communicability is high, ranging from 30% to 90%. Most cases occur in children younger than age 5 years; most deaths occur in the first year of life.

Over the last two decades, the general decline in pertussis cases in the United States began to reverse and by the late 1990s to early 2000s the incidence of disease in adolescents increased significantly. This led to the recommendation in 2005 for Tdap at age 11 and 12 years (see above) and coverage of unvaccinated 13–17-year-olds. Disease among adolescents declined. However, between 2010 and 2012 epidemics of disease in fully vaccinated (DTaP) young children (7–10-year-olds) were observed. There are several hypotheses for this lack of durable response which include waning of protection beginning after 3 years of completed vaccination and possibly changes in B pertussis antigens and genotypic characteristics. More research is required to elucidate these trends.

Despite the above trends, control of whooping cough rests mainly on adequate active immunization of all infants and those children and adults who have close contact with them.

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 is a small, gram-negative bacillus that inhabits the respiratory tracts of canines, in which it may cause “kennel cough” and 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. B bronchiseptica has a silent copy of the pertussis toxin gene. This organism possesses a β-lactamase that renders it resistant to penicillins and cephalosporins.

Concept Checks

• Bordetella species are gram-negative coccobacilli. The genus includes diverse species that range from the fastidious and virulent pathogen B pertussis, the cause of “whooping” cough, to species found primarily in animals.

• B pertussis elaborates numerous virulence factors that are responsible for pathogenesis—fimbriae and filamentous hemagglutinin promote adherence; a variety of toxins such as pertussis toxin, tracheal cytotoxin, hemolysin, and DNT mediate the severe respiratory symptoms, the hallmark lymphocytosis, and a prolonged course.

• B pertussis is fastidious and slow growing; specialized media such as Regan-Lowe agar and incubation in ambient conditions at 35–37°C for up to 7 days are required for optimum results.

• Nucleic acid amplification tests combined with culture are the diagnostic methods of choice.

• Pertussis, also known as “whooping cough,” begins with the catarrhal stage followed by the characteristic paroxysmal coughing stage that lasts weeks and ends with the convalescence stage.

• Treatment requires supportive care; erythromycin is given to reduce infectivity, but it does not alter the course of the disease. Disease is preventable by vaccination with an acellular vaccine.

• The other species of Bordetella may cause respiratory infections but are not capable of causing classic pertussis.

The brucellae are obligate parasites of animals and humans and are characteristically located intracellularly. They are relatively inactive metabolically. Brucella melitensis typically infects goats; Brucella suis, swine; Brucella abortus, cattle; and Brucella canis, dogs. Other species are found only in animals. Although named as species, DNA relatedness studies have shown there is only one species in the genus, B melitensis, with multiple biovars. The disease in humans, brucellosis (undulant fever, Malta fever), is characterized by an acute bacteremic phase followed by a chronic stage that may extend over many years and may involve many tissues.

Morphology and Identification

A. Typical Organisms

The appearance in young cultures varies from cocci to rods 1.2 μm in length, with short coccobacillary forms predominating. They are gram negative but often stain irregularly, and they are aerobic, nonmotile, and nonspore forming.

B. Culture

Small, convex, smooth colonies appear on enriched media in 2–5 days.

C. Growth Characteristics

Brucellae are adapted to an intracellular habitat, and their nutritional requirements are complex. Some strains have been cultivated on defined media containing amino acids, vitamins, salts, and glucose. Fresh specimens from animal or human sources are usually inoculated on trypticase-soy agar or blood culture media. Whereas B abortus requires 5–10% CO₂ for growth, the other three species grow in air.

Brucellae use carbohydrates but produce neither acid nor gas in amounts sufficient for classification. Catalase and oxidase are produced by the four species that infect humans. Hydrogen sulfide is produced by many strains, and nitrates are reduced to nitrites.

Brucellae are moderately sensitive to heat and acidity. They are killed in milk by pasteurization.

Antigenic Structure

Differentiation among Brucella species or biovars is made possible by their characteristic sensitivity to dyes and their production of H₂S. Few laboratories have maintained the procedures for these tests, and the brucellae are seldom placed into the traditional species. Because brucellae are hazardous in the laboratory, tests to classify them should be performed only in reference public health laboratories using appropriate biosafety precautions.

Pathogenesis and Pathology

Although each species of Brucella has a preferred host, all can infect a wide range of animals, including humans.

The common routes of infection in humans are the intestinal tract (ingestion of infected milk), mucous membranes (droplets), and skin (contact with infected tissues of animals). Cheese made from unpasteurized goats’ milk is a particularly common vehicle. The organisms progress from the portal of entry via lymphatic channels and regional lymph nodes to the thoracic duct and the bloodstream, which distributes them to the parenchymatous organs. Granulomatous nodules that may develop into abscesses form in lymphatic tissue, liver, spleen, bone marrow, and other parts of the reticuloendothelial system. In such lesions, the brucellae are principally intracellular. Osteomyelitis, meningitis, or cholecystitis also occasionally occurs. The main histologic reaction in brucellosis consists of proliferation of mononuclear cells, exudation of fibrin, coagulation necrosis, and fibrosis. The granulomas consist of epithelioid and giant cells, with central necrosis and peripheral fibrosis.

The brucellae that infect humans have apparent differences in pathogenicity. B abortus usually causes mild disease without suppurative complications; noncaseating granulomas of the reticuloendothelial system are found. B canis also causes mild disease. B suis infection tends to be chronic with suppurative lesions; caseating granulomas may be present. B melitensis infection is more acute and severe.

Persons with active brucellosis react more markedly (fever, myalgia) than normal persons to injected Brucella endotoxin. Sensitivity to endotoxin thus may play a role in pathogenesis.

Placentas and fetal membranes of cattle, swine, sheep, and goats contain erythritol, a growth factor for brucellae. The proliferation of organisms in pregnant animals leads to placentitis and abortion in these species. There is no erythritol in human placentas, and abortion is not part of Brucella infection of humans.

Clinical Findings

The incubation period ranges from 1 to 4 weeks. The onset is insidious, with malaise, fever, weakness, aches, and sweats. The fever usually rises in the afternoon; it falls during the night and is accompanied by drenching sweat. There may be gastrointestinal and nervous symptoms. Lymph nodes enlarge, and the spleen becomes palpable. Hepatitis may be accompanied by jaundice. Deep pain and disturbances of motion, particularly in vertebral bodies, suggest osteomyelitis. These symptoms of generalized Brucella infection generally subside in weeks or months, although localized lesions and symptoms may continue.

After the initial infection, a chronic stage may develop, characterized by weakness, aches and pains, low-grade fever, nervousness, and other nonspecific manifestations compatible with psychoneurotic symptoms. Brucellae cannot be isolated from the patient at this stage, but the agglutinin titer may be high. The diagnosis of “chronic brucellosis” is difficult to establish with certainty unless local lesions are present.

Diagnostic Laboratory Tests

A. Specimens

Blood should be taken for culture, biopsy material for culture (lymph nodes, bone, and so on), and serum for serologic tests.

B. Culture

Brucella agar was specifically designed to culture Brucella species bacteria. The medium is highly enriched and—in reduced form—is used primarily in cultures for anaerobic bacteria. In oxygenated form, the medium grows Brucella species bacteria very well. However, infection with Brucella species is often not suspected when cultures of a patient’s specimens are set up, and Brucella agar incubated aerobically is seldom used. Brucella species bacteria grow on commonly used media, including trypticase-soy medium with or without 5% sheep blood, brain–heart infusion medium, and chocolate agar. Blood culture media (see below) readily grow Brucella species bacteria. Liquid medium used to culture Mycobacterium tuberculosis also supports the growth of at least some strains. All cultures should be incubated in 8–10% CO₂ at 35–37°C and should be observed for 3 weeks before being discarded as being negative; liquid media cultures should be blindly subcultured during this time.

Bone marrow and blood are the specimens from which brucellae are most often isolated. The method of choice for bone marrow is to use pediatric Isolator tubes, which do not require centrifugation, with inoculation of the entire contents of the tube onto solid media. Media used in semiautomated and automated blood culture systems readily grow brucellae, usually within 1 week; however, holding the cultures for 3 weeks is recommended. Negative culture results for Brucella do not exclude the disease because brucellae can be cultivated from patients only during the acute phase of the illness or during recurrence of activity.

After a few days of incubation on agar media, the brucellae form colonies in the primary streak that are smaller than 1 mm in diameter. They are nonhemolytic. The observation of tiny gram-negative coccobacilli that are catalase positive and oxidase positive suggests Brucella species. All further work on such a culture should be done in a biologic safety cabinet. A Christensen’s urea slant should be inoculated and observed frequently. A positive urease test result is characteristic of Brucella species. B suis and some strains of B melitensis can yield a positive test result less than 5 minutes after inoculating the slant; other strains take a few hours to 24 hours. Bacteria that meet these criteria should be quickly submitted to a reference public health laboratory for presumptive identification. Brucella species are on the United States select agent list. Molecular methods have been developed to rapidly differentiate among the various biovars.

C. Serology

Laboratory diagnosis of brucellosis is most frequently accomplished by serologic testing. IgM antibody levels rise during the first week of acute illness, peak at 3 months, and may persist during chronic disease. Even with appropriate antibiotic therapy, high IgM levels may persist for up to 2 years in a small percentage of patients. IgG antibody levels rise about 3 weeks after onset of acute disease, peak at 6–8 weeks, and remain high during chronic disease. IgA levels parallel the IgG levels. The usual serologic tests may fail to detect infection with B canis because antigens used may be B abortus or B melitensis. A combination of serological tests (usually agglutination tests with nonagglutinating assays) is recommended to definitely diagnose brucellosis.

1. Agglutination test—To be reliable, serum agglutination tests must be performed with standardized heat-killed, phenolized, smooth Brucella antigens. IgG agglutinin titers above 1:80 indicate active infection. Individuals injected with cholera vaccine may develop agglutination titers to brucellae. If the serum agglutination test result is negative in patients with strong clinical evidence of Brucella infection, tests must be made for the presence of “blocking” antibodies. These can be detected by adding antihuman globulin to the antigen–serum mixture. Brucellosis agglutinins are cross-reactive with tularemia agglutinins, and tests for both diseases should be done on positive sera; usually, the titer for one disease will be much higher than that for the other.

2. Blocking antibodies—These are IgA antibodies that interfere with agglutination by IgG and IgM and cause a serologic test result to be negative in low serum dilutions (prozone), although positive in higher dilutions. These antibodies appear during the subacute stage of infection, tend to persist for many years independently of activity of infection, and are detected by the Coombs antiglobulin method.

3. Brucellacapt (Vircell, Granada, Spain)—This is a rapid immunocapture agglutination method based on the Coombs test that detects nonagglutinating IgG and IgA antibodies. It is easy to perform and has a high sensitivity and specificity. This product is not available in the United States.

4. ELISA assays—IgG, IgA, and IgM antibodies may be detected using enzyme-linked immunosorbent assays (ELISA), which use cytoplasmic proteins as antigens. These assays tend to be more sensitive and specific than the agglutination test especially in the setting of chronic disease.

Immunity

An antibody response occurs with infection, and it is probable that some resistance to subsequent attacks is produced. Immunogenic fractions from Brucella cell walls have a high phospholipid content; lysine predominates among eight amino acids; and there is no heptose (thus distinguishing the fractions from endotoxin).

Treatment

Brucellae may be susceptible to tetracyclines, rifampin, trimethoprim–sulfamethoxazole, aminoglycosides, and some quinolones. Symptomatic relief may occur within a few days after treatment with these drugs. However, because of their intracellular location, the organisms are not readily eradicated completely from the host. For best results, treatment must be prolonged. Combined treatment with a tetracycline (eg, doxycycline) and either streptomycin or gentamicin for 2–3 weeks or rifampin for 6–8 weeks is recommended. In patients with endocarditis or evidence of neurological disease, triple therapy with doxycycline, rifampin, and an aminoglycoside is suggested.

Epidemiology, Prevention, and Control

Brucellae are animal pathogens transmitted to humans by accidental contact with infected animal feces, urine, milk, or tissues. The common sources of infection for humans are unpasteurized milk, milk products, and cheese as well as occupational contact (eg, farmers, veterinarians, and slaughterhouse workers) with infected animals.

Cheese made from unpasteurized goat’s milk is a particularly common vehicle for transmission of brucellosis. Occasionally, the airborne route may be important. Because of occupational contact, Brucella infection is much more frequent in men. The majority of infections remain asymptomatic (latent).

Infection rates vary greatly with different animals and in different countries. Outside the United States, infection is more prevalent. Eradication of brucellosis in cattle can be attempted by test and slaughter, active immunization of heifers with avirulent live strain 19, or combined testing, segregation, and immunization. Cattle are examined by means of agglutination tests.

Active immunization of humans against Brucella infection is experimental. Control rests on limitation of spread and possible eradication of animal infection, pasteurization of milk and milk products, and reduction of occupational hazards wherever possible.

Concept Checks

• Brucella species are obligate intracellular pathogens found in animals; the disease in humans, brucellosis, known by a variety of synonyms, such as Malta fever, undulant fever, and so on, is caused primarily by contact with animals or animal products, especially unpasteurized milk or cheese.

• The incubation period ranges from 1 to 4 weeks; infection may begin abruptly with fever, chills, sweats, and malaise and progress to involve a multisystem illness with splenomegaly, lymphadenopathy, and osteomyelitis; chronic infection may persist for years.

• Diagnosis may be difficult and in many cases relies on serology because this fastidious organism can be difficult to cultivate even on selective media using prolonged incubation.

• Treatment consists of prolonged antimicrobial agents that are effective against intracellular pathogens such as rifampin, trimethoprim–sulfamethoxazole, fluoroquinolones, aminoglycosides, and tetracyclines.

Francisella species are widely found in animal reservoirs and aquatic environments. The taxonomy of this genus has undergone numerous changes over the years. There are seven species in the genus, the most important of which is Francisella tularensis. There are three recognized subspecies of F tularensis: tularensis (type A), holarctica (type B), and mediasiatica. Subspecies tularensis (type A) is the most virulent among this group and the most pathogenic for humans. It is associated with wild rabbits, ticks, and tabanid flies. Subspecies holarctica strains cause milder infection and are associated with hares, ticks, mosquitoes, and tabanid flies. F tularensis is transmitted to humans by biting arthropods and flies, direct contact with infected animal tissue, inhalation of aerosols, or ingestion of contaminated food or water. The clinical presentation depends on the route of infection; six major syndromes are described (see Pathogenesis and Clinical Findings).

Two other species of Francisella, Francisella philomiragia and Francisella novicida, have been associated with human disease. F philomiragia has usually been found in situations of near-drowning. These organisms will not be discussed.

Morphology and Identification

A. Typical Organisms

F tularensis is a small, gram-negative coccobacillus. It is rarely seen in smears of tissue (Figure 18-2).

B. Specimens

Blood is taken for serologic tests. The organism may be recovered in culture from lymph node aspirates, bone marrow, peripheral blood, deep tissue, and ulcer biopsies.

C. Culture

Growth requires enriched media containing cysteine. In the past, glucose-cysteine blood agar was preferred, but F tularensis grows on commercially available hemin-containing media such as chocolate agar, modified Thayer-Martin agar, and buffered charcoal yeast extract (BCYE) agar used to grow Legionella species. Media should be incubated in CO₂ at 35–37°C for 2–5 days. Caution: To avoid laboratory-acquired infections, biosafety level three (BSL III) practices are required when working with live cultures suspected of containing F tularensis. Clinical specimens require BSL II facilities and work practices.

D. Serology

All isolates are serologically identical, possessing a polysaccharide antigen and one or more protein antigens that cross-react with brucellae. However, there are two major biogroups of strains, called Jellison type A and type B. Type A occurs only in North America, is lethal for rabbits, produces severe illness in humans, ferments glycerol, and contains citrulline ureidase. Type B lacks these biochemical features, is not lethal for rabbits, produces milder disease in humans, and is isolated often from rodents or from water in Europe, Asia, and North America. Other biogroups are of low pathogenicity.

The usual antibody response consists of agglutinins developing 7–10 days after the onset of illness.

Pathogenesis and Clinical Findings

F tularensis is highly infectious: Penetration of the skin or mucous membranes or inhalation of 50 organisms can result in infection. Most commonly, organisms enter through skin abrasions. In 2–6 days, an inflammatory, ulcerating papule develops. Regional lymph nodes enlarge and may become necrotic, sometimes draining for weeks (ulceroglandular tularemia). Inhalation of an infective aerosol results in peribronchial inflammation and localized pneumonitis (pneumonic tularemia). Oculoglandular tularemia can develop when an infected finger or droplet touches the conjunctiva. Yellowish granulomatous lesions on the eyelids may be accompanied by preauricular adenopathy. The other forms of the disease are glandular tularemia (lymphadenopathy but no ulcers), oropharyngeal tularemia, and typhoidal tularemia (septicemia). All affected individuals have fever, malaise, headache, and pain in the involved region and regional lymph nodes.

Because of the highly infectious nature of F tularensis, this organism is a potential agent of bioterrorism and is currently classified on the select agent list as a Tier 1 agent. Laboratories that recover a suspected F tularensis should notify public health officials and should send the isolate to a reference laboratory capable of performing definitive identification.

Diagnostic Laboratory Tests

F tularensis may be recovered from the clinical specimens listed earlier and by performance of serologic studies. Agglutination testing either in a tubed format or in microagglutination is the standard form of testing. Paired serum samples collected 2 weeks apart can show a rise in agglutination titer. A single-tubed agglutination serum titer greater than 1:160 or a microagglutination titer of greater than 1:128 are highly suggestive if the history and physical findings are compatible with the diagnosis. Because antibodies reactive in the agglutination test for tularemia also react in the test for brucellosis, both tests should be done for positive sera; the titer for the disease affecting the patient is usually fourfold greater than that for the other disease.

Treatment

Streptomycin or gentamicin therapy for 10 days almost always produces rapid improvement. Tetracycline may be equally effective, but relapses occur more frequently. Fluoroquinolones are other potential agents. F tularensis is resistant to all β-lactam antibiotics as a result of β-lactamase production.

Prevention and Control

Humans acquire tularemia from handling infected rabbits or muskrats or from bites by an infected tick or deer fly. Less often, the source is contaminated water or food or contact with a dog or cat that has caught an infected wild animal. Avoidance is the key to prevention. The infection in wild animals cannot be controlled.

The live attenuated F tularensis vaccine (LVS) is no longer available to persons at high risk. Newer vaccines are under development.

Concept Checks

• F tularensis is a faintly staining gram-negative coccobacillus that causes the zoonotic infection tularemia that can be mediated by vectors, such as ticks, through direct contact with animals or rarely through ingestion.

• There are three subspecies of F tularensis; subspecies tularensis (type A) is the most virulent and pathogenic for humans.

• There are several clinical manifestations of tularemia depending on the type of exposure; the glandular forms are well localized and associated with less mortality than the septicemic or inhalational forms of the disease.

• Diagnosis of tularemia can be made by recovery of the organism from appropriate clinical material and by serology.

• Agents that have been useful in treatment include streptomycin, gentamicin, tetracyclines, and fluoroquinolones. Because of its virulence, F tularensis is considered a potential agent of bioterrorism.