Francisella tularensis(causative agent of tularemia): An overview

▶The History of Discovery

Tularemia causative agent was first described by G. McCoy and Ch. Chapin in Californian town Tulare in 1912. Later it was studied thoroughly by E. Francis; thereby the agent was finally named as Francisella tularensis.

▶Classification

Causative agent of tularemia, Francisella tularensis, is placed into the separate family Francisellaceae apart from other similar bacteria.

This species is further divided into 4 subspecies. Among them F. tularensis subsp. tularensis (or type A) and F. tularensis subsp. holarctica (or type B) cause the majority of cases of human infections.

F. tularensis subsp. tularensis appeared to be the most virulent agent within the genus.

▶Structure and Properties of Francisella tularensis

  • Morphology

Francisella tularensis is viewed as small (0.2-0.7 μm) pleomorphic gram-negative coccobacterium. It is a fastidious non-motile, non-spore-forming microorganism.

Within infected tissues F. tularensis produces capsule.

  • Cultivation

The bacteria don’t grow in ordinary media. They are cultured on special media with growth factors – glucose cysteine blood agar, glucose blood agar, chocolate agar or charcoal yeast extract agar at 37°C yielding small, smooth, gray-white, flat, and shiny colonies after 48 h of incubation. Increased concentrations of CO2 stimulate bacterial propagation.

Biological method is also used for microbial cultivation in laboratory animals (mice or guinea pigs).

  • Biochemical properties

Francisellas are strict aerobes, being catalase-positive, but oxidase-negative.

The bacteria ferment glucose and maltose yielding acid end products. They metabolize asparagin and produce hydrogen sulfide after protein fermenation.

There are two major biogroups of F. tularensis corresponding to bacterial subspecies – 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 enzyme.

Type В lacks these biochemical features, is not lethal for rabbits, produces milder disease in humans. Bacteria of type B are often isolated from rodents or from water in Europe, Asia, and North America.

  • Antigenic structure

F. tularensis contains somatic lipopolysaccharide O-antigen and superficial capsule-like Vi antigen.

  • Virulence factors

Virulence factors of F. tularensis are not completely studied.

These bacteria are not found to produce exotoxins.

An endotoxin is released after the degradation of microbial cells. As in brucellae, it poorly stimulates innate immune response.

Other bacterial virulent factors (e.g., capsule) inhibit phagocytosis and prevent phagosome-lysosome fusion.

Cell wall allergens stimulate the reactions of delayed hypersensitivity.

Some strains may produce hemolysins.

Francisellas are the extremely invasive bacteria, and they can infect humans even through intact skin.

  • Resistance

Francisellas are markedly resistant in the environment. They stay viable in water at 4oC for about 4 months, at 20oC – more than 2 month. Nevertheless, francisellas are sensitive to heating at 60-80oC, and commonly used disinfectants readily inactivate them.

▶Pathogenesis and Clinical Findings in Tularemia

The natural reservoirs and sources of infection of F. tularensis include numerous rodent species (rats, muskrats, mice, etc.), hares, rabbits and other animals. Humans can become infected after direct animal contact or via insect bites (ticks, biting flies, mosquitoes, etc.)

F. tularensis is highly invasive: extremely low infectious dose of 50 microbial cells penetrated through the skin or mucous membranes or even about 10 cells by inhalation is enough to result in infection. In most cases the bacteria enter the body through skin lesions.

Incubation period is short ranging 2-6 days.

The clinical manifestations of tularemia in human hosts depend on the site of entry of the bacteria (cutaneous inoculation, inhalation, or ingestion).

The infection has acute onset with chills and fever. Primary ulcerative lesion appears on the skin in the site of entry. The bacteria enter the phagocytes and actively propagate. They show remarkable resistance to microbicidal activities of phagocytes

F. tularensis replicate intracellularly causing cell destruction. Virulence factors of bacteria stimulate apoptosis of infected cells.

F. tularensis spread to regional lymph nodes that enlarge and become painful and necrotic (primary buboes). Further the bacteria migrate to organs and tissues. Degradation of bacterial cells leads to endotoxin release. Microbial accumulations in affected tissues stimulate granulomatous inflammation followed by cell-mediated reactions of delayed hypersensitivity.

Inhalation of infective aerosol results in severe pneumonitis.

Clinical forms of tularemia are classified as ulceroglandular (primarily affecting skin and lymph nodes), glandular, conjunctival and oculoglandular, oropharyngeal, pneumonic, or systemic typhoidal. Various mixed clinical variations are observed.

Severe pneumonic form of tularemia is manifested like atypical pneumonia with fever, cough with low sputum, chest pain, and ulcerative damage of lymph nodes.

Pneumonic and systemic diseases demonstrate high lethality of 30-60%; the fatality of more common local infections is about 3%.

Due to the high environmental resistance, enhanced invasiveness, minimal infectious dose, and severe course of the disease F. tularensis bacteria are accounted as the potential agents of bioterrorist attacks.

They are present in Tier 1 of US Biological Select Agents list with the highest rank of public threat.

▶Laboratory Diagnosis of Tularemia

Tularemia may be diagnosed by isolation of the bacteria from various specimens: blood, pleural fluid, sputum, lymph nodes, wounds, or gastric aspirates that depend on the clinical form of infection.

In order to avoid laboratory-acquired infection, francisella should be cultured only in biological safety cabinet of BSL-2 level maintaining all personal safety measures.

Laboratory procedures that may result in aerosol production require BSL-3 safety conditions

Microbial antigens in specimens are detected by immunofluorescence assay and ELISA.

Nucleic acids of bacteria are determined by PCR.

For cultivation cysteine blood agar or glucose blood agar are used.

More effective is biobacteriological method, where primary animal infection is followed by further inoculation of animal specimen into nutrient media for culture.

Suspected F. tularensis isolates should be delivered to a reference laboratory for confirmatory identification; it is related with the evident danger of laboratory-acquired infection.

In standard laboratory practice the diagnosis of tularemia relies largely upon the serological studies of patient’s serum for presence of specific antibodies.

Agglutination reaction is regarded as positive in a titer of 1:100-1:200 and more. Paired serum samples demonstrate the elevation of Ab titers. However, cross-reactions of antibodies with Brucella spp., Yersinia spp., etc. have been documented.

Allergic skin test with infectious allergen tularin is obviously helpful in diagnosis of infection. It evaluates specific cell-mediated response to F. tularensis based on delayed hypersensitivity.

▶Treatment and Prophylaxis of Tularemia

Chemotherapy with aminoglycosides (gentamicin or amikacin) or fluoroquinolones produces rapid clinical improvement. Tetracyclines (doxycycline) are almost equally effective.

For specific prophylaxis the individuals of high risk (e.g., laboratory personnel) are immunized with live attenuated vaccine of F. tularensis created by N. Gaisky and B. Elbert. Protection with live vaccine maintains the specific immunity for several years.