LC 14b: LEGIONELLA AND BARTONELLA

I. LEGIONELLA PNEUMOPHILA

A. History of Legionella

• 1976: A pneumonia outbreak occurred during the 58th Annual Convention of the American Legion in Philadelphia.

• 1977: Legionella pneumophila identified as the causative agent; termed Legionnaires' disease.

• Source: L. pneumophila aerosolized from contaminated water in air conditioning systems, inhaled by attendees.

• Symptoms ranged from flu-like illness to multisystem organ failure.

B. Generalities

• Classification: Gram-negative bacterium from the Legionellaceae family.

• Primary pathogen of Legionnaires’ disease, a severe form of pneumonia.

C. Morphology & Identification

C.1. Typical Organisms

• Characteristics:

  • Fastidious, aerobic, Gram-negative bacteria.

  • Length: 0.5-1 µm.

  • Often poorly stained by Gram’s method; not easily visualized in clinical specimens.

  • Smears should be prepared for suspected Legionella growth on culture media with a counterstain of 0.1% basic fuchsin.

• Staining Techniques:

  • Silver stains (Warthin-Starry and Dieterle) effective for detecting Legionellae in tissues.

C.2. Legionella Species Isolated from Humans

Figures

• Figure 1: Light micrograph of Legionella pneumophila.

• Figure 2: Gram staining of Legionella pneumophila.

• Figure 3: Direct fluorescent antibody staining of Legionella pneumophila.

D. Culture & Growth Characteristics

D.1. Culture Growth Conditions

• Media: Buffered charcoal yeast extract (BCYE) agar with ɑ-ketoglutarate, L-cysteine, iron.

• Optimal conditions: pH of 6.9, temperature of 35℃, and 90% humidity.

• Selective medium can include antibiotics; charcoal acts as a detoxifying agent.

D.2. Colony Characteristics

• Growth: Slow (visible colonies appear after approximately 3 days of incubation).

• Non-Legionella colonies noted after overnight incubation; round or flat colonies, colorless, iridescent pink or blue, translucent or speckled.

• Biochemical activity: Biochemically inert.

D.3. Confirmatory Tests

• Direct Fluorescent Antibody Test: Tests for 16SrRNA gene sequencing, employing MALDI-TOF MS.

• Catalase and Oxidase Tests: L. pneumophila is catalase positive, oxidase positive; other species may be variable in oxidase activity.

• Hippurate Hydrolysis: L. pneumophila hydrolyzes hippurate; variable presence in other Legionellae.

• Most Legionellae produce gelatinase and β-lactamase; L. micdadei does not produce these enzymes.

E. Antigens & Cell Products

• Minimum of 16 serogroups identified for L. pneumophila.

• Serogroup 1: Linked to 1976 outbreak, most common in human infections.

• Identification by serogrouping is unreliable alone due to cross-reactivity.

• Distinctive Fatty Acids: Legionellae produce unique 14-17 carbon branched chain fatty acids detectable by gas-liquid chromatography.

• Enzymes: Include proteases, phosphatase, lipase, DNAse, and RNAse.

  • Metalloprotease: Principal secretory protein with hemolytic and cytotoxic activity.

F. Pathology & Pathogenesis

• Distribution: Legionellae are found in warm, moist environments (lakes, streams).

• Environmental Resilience: Can replicate within free-living amoebas and remain in biofilms.

• Inhalation Infections: Primarily affect immunocompromised individuals; common sources include contaminated air-conditioning systems and showers.

• Pulmonary Pathology: L. pneumophila causes lobar, segmental, or patchy pulmonary infiltrations.

• Exudative Response: Involves macrophages, polymorphonuclear leukocytes (PMNs), RBCs, and proteinaceous fluid accumulation in alveoli.

• Little interstitial infiltration and minimal inflammation in bronchioles and upper airways; L. pneumophila can evade PMNs.

• Entry Mechanisms: L. pneumophila enters and thrives within human alveolar macrophages without requiring opsonization.

  • Virulence Factors:

  • Mip Protein: Central to macrophage invasion, enhancing adherence and phagocytosis.

  • Dot/Icm Type IVB Secretion System (T4SS): Facilitates translocation of over 300 effector proteins into host cells.

    • Enables the formation of a protective replicative niche inside the phagosome, circumventing lysosomal fusion.

  • Role of Transferrin Iron: Essential for microbial intracellular growth.

G. Clinical Findings

G.1. Asymptomatic Infections

• Common across all age groups.

• Clinically significant disease incidence is highest in men aged 55 and older.

G.2. Risk Factors Associated with Severe Disease

• Factors include:

  • Smoking.

  • Alcohol misuse.

  • Diabetes mellitus.

  • Chronic bronchitis/emphysema or cardiovascular diseases.

  • Immunosuppressive treatments (e.g., steroids).

  • Cancer chemotherapy.

  • Complications related to TNF-α therapy (e.g., infliximab).

H. Signs and Symptoms

• Presentation can vary:

  • Non-specific febrile illness (short duration).

  • Severe illness features high fever, chills, malaise, non-productive cough, hypoxia, diarrhea, and delirium.

H.1. Diagnostic Laboratory Tests

• Imaging: Chest radiography displays patchy and multilobar consolidation.

• Immunocompromised Patients: May present with cavitary pneumonia or pleural effusions.

• Other Lab Findings: Include leukocytosis, hyponatremia, hematuria (possibly leading to renal failure), and abnormal liver function.

I. Diagnostic Laboratory Tests

I.1. Specimen Collection

• Specimens often include expectorated sputum, bronchial washings, pleural fluid, lung biopsy samples, or blood.

I.2. Smear Results

• Legionella not demonstrable in Gram-stained smears of clinical specimens.

I.3. Culture Methods

• Culturing occurs on BCYE agar, both with and without antibiotics.

• Immunofluorescence staining for the rapid identification of cultured organisms.

• MALDI-TOF MS utilized for the expedited diagnosis of isolates.

I.4. Specific Diagnostic Tests

• Urine Antigen Test: Specific for L. pneumophila serogroup 1.

• Molecular Assays: PCR amplification for genes such as mip and 16SrRNA.

• Serologic Tests: Sensitivity ranges from 60-80% with specificity at 95-99%.

  • Antibody levels peak 4-8 weeks after infection; primarily retrospective diagnostic utility in outbreak investigations.

  • No FDA-cleared assays for Legionella detection available in the USA.

J. Immunity

• Following infection, patients generate antibodies against Legionella species.

• Antibody response peak occurs 4-8 weeks post-infection.

• Immune response encompasses both humoral and cell-mediated responses.

  • Notably, the cell-mediated immunologic response is vital for protective immunity due to the intracellular lifestyle of Legionellae.

K. Treatment

• Medications:

  • Macrolides: Examples include erythromycin, azithromycin, telithromycin, clarithromycin.

  • Quinolones: Ciprofloxacin, levofloxacin.

  • Tetracyclines: Doxycycline.

• Ineffective Drugs: β-lactams, monobactams, and aminoglycosides are generally ineffective due to β-lactamase production by many Legionellae.

• Therapy Duration: Prolonged therapy for up to 3 weeks may be necessary based on clinical scenarios.

• Discontinuation Rule: Treatment should only be halted after the patient remains afebrile for 48-72 hours.

L. Epidemiology & Control

L.1. Seasonal Trends

• Peak incidence noted during late summer to autumn.

L.2. Transmission Risks

• Travel, especially on cruise ships, increases exposure risk.

• Mechanism: Inhalation or ingestion followed by aspiration of aerosolized Legionella from contaminated water sources.

L.3. Natural Habitat

• Legionella species are found in lakes, streams, rivers, and thermally heated bodies of water.

• Growth conditions favor warm water environments, especially with amebas and bacteria.

L.4. Survival & Spread

• Legionella can exist in a sessile state, evading standard water treatment processes. Small quantities can infiltrate water distribution systems.

L.5. Control Measures

• Implementation includes hyperchlorination, superheating of water, point-of-use filters, and possible usage of copper-silver ionization.

II. BARTONELLA

A. Generalities

• Medical significance lies in three major species:

  • Bartonella bacilliformis

  • Causes: Oroya fever and Verruga peruana.

  • Bartonella quintana

  • Causes trench fever and some cases of bacillary angiomatosis.

  • Bartonella henselae

  • Causes cat-scratch disease and bacillary angiomatosis.

• Characteristics: Intracellular, Gram-negative rods that are pleomorphic, slow-growing, and difficult to isolate in laboratory settings.

• Visualization: Identified in infected tissues utilizing Warthin-Starry silver impregnation stain.

B. Bartonella bacilliformis

B.1. Stages of Infection

• Initial Stage: Oroya Fever

  • Severe infectious anemia characterized by rapid onset anemia, splenomegaly, hepatomegaly, and hemorrhage into lymph nodes.

  • Bartonellae proliferate in endothelial cells lining blood vessels, leading to potential vascular occlusion and thrombosis.

  • Diagnosis: Through blood smears and blood cultures in semi-solid media.

• Second Stage: Verruga Peruana

  • Eruptive phase commencing 2–8 weeks post-Oroya fever, featuring vascular nodular skin lesions appearing in successive crops.

  • Infection is generally self-limiting, lasting up to a year with various lesions including mucosal involvement.

  • Diagnosis: Blood cultures often yield positive results, however, anemia symptoms are absent.

B.2. Pathology & Pathogenesis

• Virulence Factors:

  • Deformin: Induces deformity of red blood cell membranes.

  • Flagella: Grant mechanical forces for invasion of RBCs; replication occurs within endocytic vacuoles facilitated by outer membrane proteins.

  • Cultured in nutrient agar supplemented with rabbit serum at 28°C for turbidity development and organism visualization.

B.3. Treatment and Control

• Available Treatments:

  • Ciprofloxacin, doxycycline, macrolides, TMP-SMX for a minimum of 10 days.

  • Parenteral therapy for those unable to take oral medications.

  • Chloramphenicol for 14 days has proven effective particularly in South America.

• Control: Focus on eliminating sandfly vectors through insecticides, repellents, and habitat disruption.

C. Bartonella henselae and Bartonella quintana

C.1. Cat Scratch Disease

• Pathology & Pathogenesis:

  • Typically presents as a benign, self-limited condition with fever and lymphadenopathy occurring 1-3 weeks post-contact with a cat (scratch, lick, or flea bite).

  • Primary skin lesions appear at sites of infection, patients may experience low-grade fever with some systemic symptoms.

  • Regional lymphadenopathy can be significant and may last weeks to months and can occasionally lead to pus discharge.

  • Potential atypical manifestations include preauricular lymphadenopathy, conjunctivitis, and more severe neurological signs and symptoms.

C.2. Diagnosis

• Diagnosis Indicators:

  • Clinical history, lymph node aspirate (often without culturable bacteria), and characteristic histopathological findings (granulomas with detectable bacteria via silver staining).

  • A positive skin test can aid diagnosis; indirect fluorescent antibody tests with titer >1:64 strongly supports infection.

• Culture Method: Not generally recommended due to difficulty isolating B. henselae.

C.3. Treatment

• Supportive care is standard for most cases; may include hot soaks and analgesics.

• Surgical options to drain any excessively enlarged lymph nodes may help relieve symptoms.

• While tetracycline, azithromycin, TMP-SMX, rifampin, gentamicin, and fluoroquinolone may have anecdotal efficacy, recent studies do not consistently support antibiotic treatment.

C.4. Bacillary Angiomatosis

• Pathology & Pathogenesis:

  • Affects primarily immunosuppressed individuals (e.g., HIV/AIDS).

  • Presents with nodular lesions characterized by capillary proliferation and commonly occurs in various organs alongside systemic symptoms such as fever and weight loss.

• Diagnosis:

  • Confirmed through characteristic histopathologic findings and the presence of pleomorphic bacilli in silver-stained tissues.

  • Isolation possible via direct culture from biopsy of affected tissues.

C.5. Trench Fever

• Symptomology: Characterized by sudden fever onset accompanied by headache, malaise, restlessness, and leg pain.

• Fevers coincide with B. quintana release into the bloodstream every 3-5 days, with individual episodes lasting around 5 days.

C.6. Reservoirs and Transmission

• Reservoirs:

  • B. henselae primarily infects domestic cats.

  • B. quintana is associated with humans and body lice.

References

• Velasco, L. (2025). LEGIONELLA PNEUMOPHILIA. [PPT & Lecture].

• Brooks, G. F., Jawetz, E., Melnick, J. L., & Adelberg, E. A. (2019). Jawetz, Melnick & Adelberg's Medical Microbiology (28th ed.). McGraw-Hill Medical.