Bacte Lec 15: Miscellaneous Gram-Negative Bacilli

Capnocytophaga

it is a normal microbiota of the oral cavity of humans

Septicemia

What disease does Capnocytophaga cause in patients with neutropenia?

Fusobacterium spp

what org does Capnocytophaga resemble? (thin and often fusiform)

gliding motility

even though Capnocytophaga doesn’t have flagella, it produces what?

sucrose, glucose, maltose, lactose

what does Capnocytophaga ferment?

negative

Is Capnocytophaga positive or negative in indole test?

Blood cultures from patients who have neutropenia with oral ulcers

Soft tissue infection

Peritonitis

Endocarditis

What are the common site of clinical isolation of Capnocytophaga?

Capnocytophaga ocracea

It is the most common isolate (of the Capnocytophaga spp)

Capnocytophaga canimorsus

Capnocytophaga cynodegmi

these are normal inhabitants of the oral cavity of dogs & cats; infection from dog/cat bite

Legionella

it is a non-spore forming, thin, gram-negative bacilli; faintly staining

environment (esp water)

where can Legionella be found?

Chlorine concentrations of 3 mg/L

What can Legionella tolerate?

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

What is the mode of transmission of Legionella?

Hot water systems, cooling towers, evaporative condensers

What are the major reservoirs of Legionella?

Epidemic of pneumonia; Pennsylvania American Legion

When was Legionella considered to cause human disease?

Legionella pneumophila

What caused the epidemic of pneumonia among the members of Pennsylvania American Legion?

• Ability to exist as intracellular pathogens (amebae & mammalian cells)

• Survive inside phagosomes, preventing the formation of phagolysosome

• The ability to multiply at temperature range of 30°C - 43°C and survive for varying periods at 40°C - 60°C

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

What are the virulence factors of Legionella?

Legionnaire’s Disease

It is a febrile disease with pneumonia

Pneumonia

The predominant form of Legionnaire’s Disease

legionnaire’s Disease

community acquired bacterial pneumonia

2-10 days

Incubation period of Legionnaire’s disease

• Nonproductive cough

• fever

• headache

• Myalgia

• Sputum ay be bloody or purulent

• May lead to extrapulmonary infection

symptoms of Legionnaire’s Disease

Pontiac fever

Influenza-like febrile disease

Pontiac fever

Non-pneumonic form of legionellosis

inhalation of bacterial toxins or an acute allergic reaction to bacteria

causes of Pontiac fever

2 days

incubation period of Pontiac Fever

• Fever

• headache

• Myalgia (lasts 2-5 days; subside without medical intervention)

symptoms of Pontiac fever

Legionnaire’s Disease

Lab Diagnosis:

combination of culture and urine antigen detection

Pontiac fever

Lab Diagnosis:

Serology

Specimens for culture and direct examination

Lab Diagnosis:

sputum, bronchoalveolar lavage, and bronchial washings

other tissues or fluids (e.g. pleural fluid)

Lab diagnosis that is generally acceptable when suspicion is high (Specimen for culture and direct examination)

freezing the specimens at -70°C

done if processing will be delayed for several days

Pleomorphic, weakly staining, gram-negative bacilli

found outside of and within macrophages and segmented neutrophils

Diff-Quik and Giemsa

stains used for microscopic examination of Legionella

Buffered Charcoal Yeast Extract (BCYE) Agar with L-cysteine

best culture media for Legionella isolation

Grayish white or blue-green convex, and glistening; approximately 2-4mm in diameter

appearance of Legionella colonies in BCYEA

“ground-glass” appearance, light gray and granular

appearance of the central portion of young colonies in BCYEA

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

appearance of the periphery of the colony in BCYEA

polymyxin B, anisomycin, & either vancomycin or cefamandole

Selective BCYEA contains:

Incubate at 35°C in air; increased CO2 enhances growth of some of the more fastidious spp

Incubation requirement of BCYEA

Fastidious, aerobic bacteria

do not grow on SBA and require L-cysteine for growth

Acid treatment

the pre-treatment before inoculation

treating non-sterile specimen; it kills bacteria so only Legionella will remain

Purpose of the pre-treatment

1:10 with 0.2N potassium chloride - hydrochloric acid

dilution of the aliquot of the specimen

1:10 tryptic soy broth or distilled water

specimens from normally sterile sites should be diluted

to dilute microbial inhibitors (e.g. complement antigen and antimicrobial agents)

purpose of diluting 1:10 tryptic soy broth or distilled water

Bordetella pertussis

cases whooping cough

Whooping cough or Pertussis

highly contagious, acute infection of the upper respiratory tract

Person to person through inhalation of respiratory droplets

Mode of transmission of whooping cough

• Catarrhal Stage

• Parocysmal stage

• Convalescent phase

Stages of infrection (whooping cough)

Catarrhal Stage

Symptoms are the same as for a mild cold with runny nose and mild cough; lasts several weeks

Parocysmal Stage

Severe and violent coughing; lasts 1-4 weeks

15-25 paroxysmal coughing episodes

Paroxysmal coughing episodes occur in 24hrs

Whooping

Result of air rapidly inspired into the lungs past the swollen glottis

Convalescent phase

Begins within 4 weeks of onset with decrease in frequency and severity of the coughing spells; cause mild illness and symptomatic infection

• Fimbriae (FIM) Type 2 and 3

• Filamentous hemagglutinin (FHA)

• Pertactin (PRN)

• Tracheal Colonization Factor

Function: Adhesion

Fimbriae (FIM) Type 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

Tracheal Colonization Factor

• Pertussis toxin

• Adenylate cyclase toxin

• Dermonecrotic toxin

• Tracheal cytotoxin

• Endotoxin

• Type III secretion

Function: Toxicity

Pertussis toxin ( encoded by the ptx gene, an A/B toxin related to cholera toxin)

Induces lymphocytosis and suppresses chemotaxis and oxidative responses in neutrophils and macrophages

Adenylate Cyclase Toxin

Hemolyzes red cells and activates cyclic adenosine monophosphate, thereby inactivating several types of host immune cells

Dermonecrotic Toxin

Exact role is unknown

Tracheal cytotoxin

Ciliary dysfunction and damage

Endotoxin

Lipopolysaccharide

Type III secretion

• Outer membrane

• Siderophore production

Function: Overcome host defenses

Outer membrane

Inhibits host lysozyme

Siderophore production

Prevents host lactoferrin and transferrin from limiting iron

Nasopharyngeal aspirates or a nasopharyngeal swab (calcium-alginate or Dacron on a wire handle)

Specimen for Bordetella Lab Diagnosis

• throat

• sputum

• anterior nose

Specimens from these 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:

Half-strength Regan-Lowe agar

enhances recovery when used as a transport and enrichment medium

• 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

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)

Stainer-Scholte

Synthetic agar lacking blood products

Plates are incubated at 35C in a humidified atmosphere without elevated carbon dioxide for up to 12 days

Incubation requirement of the culture media of Bordetella

mercury drops

Young colonies of B. pertussis and B. parapertussis

whitish gray with age

Colonies become (what color)

Erythromycin

Drug of choice for pertussis

Pasteurella

Zoonotic disease; Normal flora of oral cavity in birds and mammals

• Septicemia

• Arthritis

• Endocarditis

• Osteomyelitis

• Meningitis

• Soft tissue (cutaneous) infection from animal bites

Pasteurella causes:

Pasteurella multocida

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

Pasteurella multocida

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

Modes of Transmission of Pasteurella multocida

Pasteurella canis

dogs; Gram-negative coccobacilli; Non-motile; Facultative anaerobe

SBA and CHOC agar

Pasteurella grow on