Miscellaneous Gram-negative Bacilli

Legionella

• Non-spore forming, thin, gram-negative bacilli

• Faintly staining

• Found in the environment especially water

• Tolerate chlorine concentrations of 3 mg/L

exposure to contaminated water (faucets, showerheads, public fountains, aircon)

Mode of Transmission of Legionella

Hot water systems, cooling towers, and evaporative condensers are major reservoirs

Reservoirs of Legionella

Legionella

First recognized to cause human disease during an epidemic of pneumonia that occurred among members of the Pennsylvania American Legion who had gathered in Philadelphia to celebrate the 1976 bicentennial

Capnocytophaga

• normal microbiota of the oral cavity

• fastidious, facultative anaerobe

• thin and often fusiform

• can produce gliding motility on slid surfaces

• ferment sucrose, glucose, maltose, and lactose

• indole negative

• Blood cultures from patients who have neutropenia with oral ulcers

• Soft tissue secretions

• Peritonitis

• Endocarditis

Common site of infection of Capnocytophaga

Capnocytophaga ocracea

Most common clinical isolate of Capnocytophaga

Capnocytophaga canimorsus

Normal inhabitants of the oral cavity of dogs and cats

Capnocytophaga cynodegmi

Infection from dog or cat bite

Legionella pneumophila

The epidemic of pneumonia associated with Legionella is caused by what species?

Legionella

Ability to exist as intracellular pathogens (amebae and mammalian cells)

Legionella

Survive inside phagosomes, prevent the formation of phagolysosome

Legionella

The ability to multiply over the temperature range of 20C to 43C and survive for varying periods at 40C to 60C

Legionella

Capacity to adhere to pipes, rubber, plastics, and sediment and persist in piped water systems, even when flushed.

Legionnaire’s Disease

• Febrile disease with pneumonia

• Predominant manifestation: pneumonia

• Community acquired bacterial pneumonia

2-10 days

Incubation period of Legionnaire’s disease

• Nonproductive cough

• Fever

• Headache

• Myalgia

• Sputum may be bloody or purulent

• May lead to extrapulmonary infection

Symptoms of Legionnaire’s disease

Pontiac fever

• Influenza-like febrile disease

• Non-pneumonic form of legionellosis

• Inhalation of bacterial toxins or an acute allergic reaction to the bacteria

• Previously healthy individuals who complain of flu-like symptoms

• Legionnaire’s disease

• Pontiac fever

• Asymptomatic infection

Infections caused by Legionella

Combination of culture and urine antigen detection

Laboratory diagnosis for Legionnaire’s disease

Serology

Laboratory diagnosis for Pontiac fever

Sputum, bronchoalveolar lavage, and bronchial washings

Specimen for Culture and direct examination for Legionella

Other tissue fluids (pleural fluid)

Specimen that are generally acceptable for culture and direct examination of Legionella when suspicion is high

Pleomorphic, weakly staining, gram-negative bacilli found outside of and within macrophages and segmented neutrophils

in microscopic examination of Shigella, what does it look like?

-70 degrees Celsius

If specimen for Legionella will be delayed for several days, it should be freeze at what temperature?

Diff-Quick and Giemsa stain

Other stains that can be used for Legionella

BCYE (Buffered charcoal yeast extract) agar with L- cysteine

Best culture media for Legionella isolation

grayish white or blue-green, convex, and glistening

In BCYE agar, what does the colonies of Legionella look like?

2 to 4 mm

colonies of Legionella in BCYE measures approximately?

ground glass appearance, light gray, and granular

Central portion of young colonies of Legionella in BCYE has a?

polymyxin B, anisomycin, and either vancomycin or cefamandole

Selective BCYE agar contains?

Incubate at 35C in air; increased CO2

What environmental requirements can enhance the growth of some of the more fastidious species of Legionella?

pink or light blue or bottle green bands with a furrowed appearance

The periphery colony of Legionella in BCYE has what appearance?

Acid treatment

TRUE OR FALSE: In isolation methods of Legionella, what should be done to the specimen before inoculation?

1:10 with 0.2N potassium chloride – hydrochloric acid

In Legionella, aliquot of the specimen is first diluted to and with?

1:10 in tryptic soy broth or distilled water

For Legionella: Specimens from normally sterile sites should be diluted

Whooping cough or Pertussis

• Transmission occurs person to person through inhalation of respiratory droplets

• Highly contagious, acute infection of the upper respiratory tract

• caused by Bordetella pertussis

Catarrhal stage

• Symptoms are the same as for a mild cold with a runny nose and mild cough

• Lasts several weeks

Paroxysmal stage

• Severe and violent coughing

• 15 to 25 paroxysmal coughing episodes can occur in 24 hours

• Vomiting and “whooping” (the result of air rapidly inspired into the lungs past the swollen glottis)

• Lasts 1-4 weeks

Convalescent phase

• Begins within 4 weeks of onset with a decrease in frequency and severity of the coughing spells

• Cause mild illness and symptomatic infection, primarily in household contacts and in a number of unvaccinated and previously vaccinated children

• Fimbriae (FIM) type 2 and 3

• Filamentous hemagglutinin (FHA)

• Pertactin (PRN)

• Tracheal colonization factor

• Pertussis toxin

• Adenylate cyclase toxin

• Dermonecrotic toxin

• Tracheal cytotoxin

• Endotoxin

• Type III secretion

• Outer membrane

• Siderophore production

Major virulence determinants of Bordetella pertussis

Adhesion (auto transporters)

• Fimbriae (FIM) type 2 and 3

• Filamentous hemagglutinin (FHA)

• Pertactin (PRN)

• Tracheal colonization factor

What is the function of these determinants (B. pertussis)?

Toxicity

• Pertussis toxin

• Adenylate cyclase toxin

• Dermonecrotic toxin

• Tracheal cytotoxin

• Endotoxin

• Type III secretion

What is the function of these determinants (B. pertussis)?

Overcome host defenses

• Outer membrane

• Siderophore production

What are the function of these determinants (B. pertussis)?

Fimbriae (FIM), types 2 and 3

Serotype-specific agglutinins for colonization of respiratory mucosa

Filamentous hemagglutinin (FHA)

Mediates adhesion to the ciliated upper respiratory tract

Pertactin (PRN)

Mediates eukaryotic cell binding and is highly immunogenic

Pertussis toxin

Induces lymphocytosis and suppresses chemotaxis and oxidative responses in neutrophils and

macrophages

ptx gene

pertussis toxin is encoded by?

ptx gene

an A/B toxin related to cholera toxin

Adenylate cyclase toxin

Hemolyzes red cells and activates cyclic adenosine monophosphate, thereby inactivating

several types of host immune cells

Tracheal cytotoxin

Ciliary dysfunction and damage

Outer membrane

Inhibits host lysozyme

Siderophore production

Prevents host lactoferrin and transferrin from limiting iron

Nasopharyngeal aspirates or a nasopharyngeal swab

Specimen for Bordetella

calcium-alginate or Dacron on a wire handle

swab used for the laboratory diagnosis of Bordetella

TRUE

TRUE OR FALSE: For Bordetella, specimens obtained from the throat, sputum, or

anterior nose are not accepted because these sites are not aligned with ciliated epithelium

• Half-strength Regan-Lowe agar

• Cold casein hydrolysate medium

• Casamino acid broth

Transport media that can be used for Bordetella

Half-strength Regan-Lowe agar

enhances recovery when used as a transport and enrichment medium for Bordetella

• Bordet-Gengou

• Modified Jones-Kendrick charcoal

• Regan-Lowe

• Stainer-Scholte

Culture media used for Bordetella

Bordet-Gengou

Potato infusion agar with glycerol and sheep blood with methicillin or cephalexin (short shelf-life)

Cephalexin

is superior to methicillin and penicillin for inhibiting normal respiratory flora

Modified Jones-Kendrick charcoal

Charcoal agar with yeast extract, starch, and 40 ug cephalexin (2- to-3-month shelf-life but inferior to Regan-Lowe agar)

Regan-Lowe

• Best for the recovery of B. pertussis from nasopharyngeal swabs

• Charcoal agar with 10% horse blood and cephalexin (4- to 8- week-shelf-life)

Erythromycin

Drug of choice for pertussis

Pasteurella

• Zoonotic

• Normal flora of oral cavity in birds and mammals

• Soft tissue (cutaneous) infection from animal bites

• Septicemia

• Arthritis

• Endocarditis

• Osteomyelitis

• Meningitis

Pasteurella causes?

Pasteurella multocida

• Commensal found in nasopharynx and gastrointestinal tract of wild and domestic animals

• Potential upper respiratory commensal in humans having extensive occupational exposure to animals

• Bite or scratch from variety of veterinary hosts (usually feline or canine)

• Infections may be associated with non-bite exposure to animals

• Less commonly, infections may occur without history of animal exposure

Mode of transmission of Pasteurella multocida

Pasteurella cannis

• Dogs

• Gram-negative coccobacilli

• Non-motile

• Facultative anaerobic coccobacilli

• Bipolar staining: “Safety pin” appearance

Chocolate agar and Sheep’s Blood Agar

Pasteurella grows on what agar?

nonhemolytic colonies that may appear mucoid

what is the colonies of Pasteurella on SBA?

24 hours of incubation at 37C

colonies of Pasteurella may appear mucoid after?

graying colonies

Colonies of Pasteurella

narrow green-to-brown halo

what is produced around the colonies of Pasteurella after 48 hours?

Brucella

• Small, facultative, intracellular, nonmotile, aerobic, gram-negative coccobacilli or short rods

• Capable of surviving in soil, aborted fetuses, bovine stoon, milk and ice cream, and fresh cheese for extended periods

• A cause of devastating economic loss among domestic livestock

• Considered category B select biological agents by the CDC

10 weeks

Brucella can live in soil for how many weeks?

11 weeks

Brucella can live in Aborted fetuses for how many weeks?

17 weeks

Brucella can live in Bovine stoon for how many weeks?

3 weeks

Brucella can live in milk and ice cream for how many weeks?

several months

Brucella can live in fresh cheese for how many weeks?

• Mediterranean and Persian Gulf countries

• India

• parts of Mexico

• Central and South America

Brucella occurs worldwide, especially in?

• Ingestion of infected unpasteurized animal milk products

• Inhalation of infected aerosolized particles

• Direct contact with infected animal parts through rupture of skin and mucous membranes

• Accidental inoculation of mucous membranes by aerosolization

• Systemic, deep-seated disease resulting in various long-term sequelae

Mode of transmission of Brucella

Ingestion of infected unpasteurized animal milk products

most common means of transmission of Brucella

Inhalation of infected aerosolized particles

laboratory-acquired infection, the most important source of transmission of Brucella:

Brucella abortus

Brucella species wherein the preferred animal host is cattle

Brucella melitensis

Brucella species wherein the preferred animal host are sheep or goats

Brucella suis

Brucella species wherein the preferred animal host is swine

Brucella canis

Brucella species wherein the preferred animal host are dogs

Brucella ovis

• Brucella species wherein the preferred animal host are rams

• not associated with human infection

Brucella neotomae

• Brucella species wherein the preferred animal host is desert and wood rats

• Not associated with human infections

• Blood

• Blood marrow

• Cerebrospinal fluid (CSF)

• Pleural and synovial fluids

• Urine

• Abscesses

• Other tissues

Specimen used for Brucella

Blood agar, Chocolate agar, Brucella agar on infusion base

Culture media used for Brucella

Brucella agar on infusion base

for Brucella, this is recommended for specimen types other than blood

5% heated horse or rabbit serum

What is added to the culture media used for Brucella to enhance its growth?

appear small, convex, smooth, translucent, nonhemolytic, and slightly yellow

Colonies of Brucella

Small coccobacilli that resemble fine grains of sand

Gram stain of Brucella

• oxidase

• catalase

• urease

What biochemical tests is Brucella positive?

Francisella

• Facultative

• Intracellular pathogens

• Nonmotile

• Non-spore forming

• Strict aerobes

cysteine, cystine, or another sulfhydryl and a source of iron

Francisella requires what for enhanced growth?