Brucella, Brucella, Francisella,Francisella, Bordetella, Bordetella,

Brucella, Brucella, Francisella,Francisella, Bordetella, Bordetella,Fastidious Gram-Fastidious Gram-Negative RodsNegative Rods

Genera of Uncertain Affliation • Gram(-), small,

coccobacilli• Strict aerobe,

nonfermenter• Most nonmotile• Fastidious, enriched

media for culture• Slow culture growth,

prolong incubation• Brucella - brucellosis• Francisella - tularemia• Bordetella – pertussis,

important pediatric disease

Brucella: Zoonotic Pathogen

• Worldwide - >500,000 cases/year• USA - <100 cases/year, control disease

in animals• Usually mild, AS disease in natural

animal host• Infect animal tissue rich in erythritol;

localize infection of breast, uterus, placenta, fetus, epididymus

• Intracellular parasite of macrophage• Cause of sterility, abortion, AS carriage

of infected animals

Brucella: Genera • Four species infect humans – wide

disease spectrum:– B. melitensis (goats, sheep) - most common

cause of brucellosis, causes severe disease– B. suis (swine) – destructive lesions,

prolonged disease– B. abortus (cattle) – mild disease– B. canis (dog) – mild disease

• DNA studies show one species – B. melitensis, others are biovars

Brucella: Lab Culture• Brucella Blood agar - fastidious,

requires enriched media, CBA containing meat infusion or tryptone

• Growth on CBA or chocolate agar, slow and may take 72 hours

• Colonies start as tiny pinpoint, translucent colonies, become gray with age

• Most clinical isolates obtained from blood culture

Brucella: Lab ID• Collect patient blood specimen, inoculate

directly into blood culture bottle• Serum serology for Brucella antibodies to

test for infection • Extreme care required for safe handling

of specimen and culture in lab• Biosafety Level-3 pathogen - transmitted

by respiratory route, produce serious disease; handle in biological safety cabinet

Brucella melitensis: Brucellosis (Undulant Fever)

• “Malta (Island of Melita) Fever”• In humans, ingestion of unpasteurized

milk or cheese from goats, cows; also acquire MO via direct contact with infected animals, inhalation

• At risk are veterinarians, slaughterhouse workers, farmers, lab microbiologists

• CDC category B Select Biological Agent– Easy to disseminate– Moderate morbidity, low mortality

Brucellosis (Undulant Fever) • Localized abcesses at site on inoculation;

bacteremia, MO phagocytized; localized in RES tissues (spleen, liver, bone marrow, lymph nodes, kidneys)

• Infection ranges from:– Asymptomatic, subclinical– Subacute - malaise, chills, sweats, fatigue, myalgia,

weight loss, nonproductive cough– Acute infection – fever, chills, endotoxic disease

• Fever in all patients and intermittant:– Typically spikes each evening– Coincides with release of MO from phagocytes– “Undulant” fever

Brucella: Treatment and Prevention

• Chemotherapy difficult, intracellular survival of MO in macrophages

• Combination doxycycline + rifampin• Tetracycline toxic for pregnant moms and

young kids (<8 yr.) - SXT drug of choice• Control by ID (serology) infected animals:

– Eliminate infected herds– Animal vaccine to prevent infection

• Pasteurization of dairy products kills MO• Biohazard safety precautions in the lab

Francisella tularensis: Zoonotic Pathogen

• Tulare county – infected rodent transmit to humans by tick bite

• Tularemia – glandular fever, rabbit fever, tick fever, deerfly fever

• Worldwide; USA - low incidence ~100 cases/year

• Intracellular parasite – survive prolonged periods in macrophage of RES (lymph nodes, bone marrow, spleen, liver, kidneys)

F. tularensis: Lab Culture • Fastidious, require enriched

media• Chocolate, MTM + antimicrobials• BCYE (buffered charcoal-yeast

extract) – selective media for Legionella

• Colonies may grow in 24 hours, or 5-7 days

• Specimen – ulcer scrape, lymph node biopsy, sputum

• Collection and processing specimen extremely hazardous:– MO highly contagious (<100 MO)– Penetrate skin and mucous

membranes– Inhale aerosols

F. tularensis: Lab ID • Best done by serology:

– No previous infection - antibody titer ≥1:160

– Patient with previous antibody - 4x increase in paired serum (acute, convalescent)

• Culture done under biological safety cabinet (Biosafety Level-3 pathogen)

• Laboratory infection common

Tularemia: Transmission • MO in wild animals, domestic animals,

birds, fish, ticks, water• Acquired by:

– Direct contact infected animals– Ingestion contaminated meat or water– Inhalation

• Infectious dose low ~10 • A disease mostly in rabbits, rodents• Most often transmitted to human by

ticks or deer flies fed on infected rodents, or pet catching infected rabbit

Tularemia: Disease (Mode of Entry)

• 1) Ulceroglandular – through skin abrasions:– After ~48 hours, lesion occurs

at inoculated site, forms ulcer– Headache, pain, fever,

adjacent lymph nodes enlarged

– If not contained - progress to septicemia, pneumonia, abscesses throughout body

– MO survives long periods of time inside phagocytic cells

Tularemia: Disease • 2) Typhoidal – ingestion

– Focus of infection in mouth, throat, GI tract

– Systemic signs of fever, toxemia in liver, spleen

• 3) Pneumonic – inhalation– Most severe form of

disease– Pneumonia with high

mortality rate (30%) if untreated

F. tularensis: Bioterrorism Agent

• CDC category A Select Biological Agent• Risk to national security• Spread person-to-person, or easily

disseminated• Result in high mortality• Lead to great public health impact and

public panic

F. tularensis: Treatment and Prevention

• Streptomycin drug of choice, however not readily available, high toxcity

• Gentamycin good alternative• Attenuated, live vaccine protects

against inhalation form of disease; for at risk (farmer, hunter, veterinarian, lab microbiologist)

• Avoid reservoirs and vectors of infection

• Wear protective clothing, use insect repellant (DEET) to reduce risk of tick, fly bite

Case Study 5 - Francisella• A 27-year-old man was mowing his field

when he ran over two young rabbits.• When he stopped his mower, he realized

that two other rabbits were dead in the unmowed part of the lawn.

• He removed all the rabbits and buried them.

• Three days later he developed a fever, muscle aches, and a dry, nonproductive cough.

• Over the next 12 hours he got progressively sicker and was transported by his wife to the area hospital.

Case Study 5 - Francisella• Results of a chest x-ray showed

infiltrates in both lung fields.• Blood cultures and respiratory

secretions were collected, and antibiotics were initiated.

• Blood cultures became positive with small gram-negative rods after 3 days of incubation, and the same organism grew from the respiratory specimen that was incubated onto BCYE agar.

Case Study - Questions• 1. What test should be performed to

confirm the tentative diagnosis of Francisella tularensis?

• 2. This infection was presumably acquired by inhalation of aerosolized contaminated blood. What are the most common sources of F. tularensis infections and the most common routes of exposure?

• 3. What are the different clinical manifestations of F. tularensis?

Bordetella: Pertussis• Endemic disease worldwide - >60 million

cases annually, >200,000 deaths• Incidence reduced with Diphtheria,

Pertussis, Tetanus vaccine (DPT)• Pertussis in USA:

– Inadequately immunized children (5 doses)– Child not vaccinated due to parental fear of

vaccine-related neurologic toxcity– Increase infections in adolescents and adults

(reservoir) due to waning immunity, no booster vaccination

– Recent resurgence and outbreaks estimated ~3 million

Bordetella: Genera• Three medically important species:

– B. pertussis – “severe cough”, pertussis (whooping cough) in humans

– B. parapertussis – “resembling”, milder pertussis in humans

– B. bronchoseptica – “trachea, septic”, respiratory disease in animals; occasional respiratory or wound infection in humans

Bordetella pertussis: Lab Culture

• No growth on common lab media:– Additional nutrients needed– Use of adsorbents to remove media

substances (fatty acids, sulfides) toxic to B. pertussis

• Bordet-Gengou media – glycerol, potato infusion, albumin (binds fatty acids), up to 50% sheep RBCs

• Charcoal agar supplemented with 10% horse blood, with or without cephalexin

• Usually 3-7 days for growth, 370 C

B. pertussis: Virulence Factors - Toxins

• Pertussis toxin (PT):– Increase in cAMP; increase respiratory

secrections, mucus leads to coughing stage of disease

• Adenylate cyclase/hemolysin toxin:– Increase in cAMP; inhibits phagocytes– Protect MO during infection, early stages of

disease• Tracheal cytotoxin:

– Due to bacterial peptidoglycan– Killing, sloughing of ciliated cells– Interferes with cell DNA synthesis, repair,

regeneration– Disrupts normal clearance in RT– Damage leads to “whooping” cough

B. pertussis: Virulence Factors (Cell Surface)

• Filamentous hemagglutinin (FHA) – attaches to host cell cilia, antibody made against it protective (used for acellular vaccine)

• Pertactin – adhesion, antibody made against it protective (used for acellular vaccine)

• Lipooligosaccharide - surface of the bacteria, potent endotoxin activity

B. pertussis: Whooping Cough• Infection by inhalation of infected

droplets or direct contact with secretions• Highly infectious, 2º attack rate = 80%• MO attaches to RT ciliated cells • Incubation 1-2 weeks, MO multiplies

liberates toxins

Whooping Cough: Three Stages

• 1) Catarrhal stage:– Resembles cold, mild cough and sneezing– Large number MO spread through

respiratory secretions– Last ~2 weeks

• 2) Paroxysmal stage:– Rapid, consecutive coughs, rapid intake of

air (whooping sound)– 40-50 daily paroxysms; vomiting,

exhaustion– Last 3-4 weeks

• 3) Convalescent stage:– Symptoms gradually subside– Last 3-4 weeks– Complete recovery may need months

Whooping Cough • B. pertussis rarely spreads to other

sites, but lot of cell damage may occur:– Local tissue of RT– Systemic damage by spread of exotoxin

• Secondary complications may occur– Pneumonia– Otitis media– CNS dysfunction - seizures, encephalopathy

occurs ~10% cases; unknown cause

B. pertussis: Treatment and Prevention

• Primarily supportive care• Convalescence correlated with regeneration of

ciliated epithelium• Erythromycin:

– Effective early stage of disease before toxins– Reduces infectivity of patient

• DTaP vaccine – subunit acellular pertussis:– PT toxoid, FHA hemagglutinnin, Pertactin– Lower incidence of side-effects– Children: five doses – 2, 4, 6, 18 months; booster at 4-6

years– Adolescents, adults: boosters – 12 years; 19-65 years

Class Assignment• Textbook Reading:• Chapter 18 Haemophilus and Other Fastdious

Gram-Negative Rods - Brucella, Francisella

• Chapter 40 Zoonotic Diseases – Tularemia, Brucellosis (pgs. 941-943)

• Chapter 32 Upper and Lower RTI – Pertussis (pgs. 782-783)

• Key Terms• Learning Assessment Questions

Case Study 6 - Bacillus• A 56-year-old female postal worker

sought medical care for fever, diarrhea, and vomiting.

• She was offered symptomatic treatment and discharged from the community hospital emergency department.

• Five days later she returned to the hospital with complaints of chills, dry cough, and pleuritic chest pain.

• A chest radiograph showed a small right infiltrate and bilateral effusions but no evidence of a widened mediastinum.

Case Study 6 - Bacillus• She was admitted to the hospital, and

the next day her respiratory status and pleural effusions worsened.

• A computerized tomographic (CT) scan of her chest revealed enlarged mediastinial and cervical lymph nodes.

• Pleural fluid and blood was collected for culture and was positive within 10 hours for gram-positive rods in long chains.

Case Study - Questions• 1. The clinical impression is that this

woman has inhalation anthrax. What tests should be performed to confirm the identification of the isolate?

• 2. What are the three primary virulence factors found in B. anthracis?

• 3. Describe the mechanisms of action of the toxins produced by B. anthracis.

• 4. Describe the two forms of B. cereus food poisoning. What toxin is responsible for each form? Why is the clinical presentation of these two diseases different?