Week 5 - Fastidious Gram Negative Rods

Fastidious Gram Negative Rods Introduction:

• Fastidious organisms have complex nutritional requirements, and do not grow readily on routinely used media.

• Many require the addition of specific nutritional growth factors to culture medium to make isolation and identification possible

• Depending on Genus, fastidious GNR can be facultatively anaerobic (grows in presence or absence of oxygen), while others are strict aerobes or microaerophilic.

• Glucose fermenter non-Enterobacteriaceae

• Greatly varied in natural habitats and sources of infection

• Human infections with fastidious GNR relatively uncommon.

• Most commonly recovered organisms include: Haemophilus influenzae, Francisella tularensis, Legionella pneumophila, and Bordetella pertussis

• Other organisms discussed in this lecture include: Brucella, the cause of brucellosis (and potential bioterrorism agent); the HACEK group of organisms, which are implicated in sub acute bacterial endocarditis;

  Streptobacillus, a slow growing agent of rat-bite fever; as well as misc fastidious including Bartonella, Pasteurella & Capnocytophaga species.

Haemophilus spp: General Characteristics:

• Slow growing Facultative anaerobes

• Usually require enriched medium that contains fresh blood or some of it’s components

• Most need Heme (X factor) or NAD (V factor) for growth

• Factor X can diffuse from intact red blood cells and is available in sheep blood agar; Factor V cannot diffuse from intact RBCs and is only released due to hemolysis

• Chocolate agar is recommended growth medium; due to fact that it is high in concentration for both factors because sheep blood is added to hot liquid agar causing lysis of the red cells and release of the factors

• Some require 5-10% CO2

• Short, pleomorphic (many forms) GNR

  • Sometimes assume coccobacillary morphology or cocci-like appearance on Gram stain

How do you acquire Francisella Tularensis:

• Enters the respiratory tract and (2) the lamina propria of the respiratory bronchioles via M cells; (3) Digested antigen is taken up by dendritic cells; the dendritic cells travel to regional lymph nodes and present F. tularensis antigens to T-helper 1 cells; (4) T-helper 1 cells proliferate; they may return to site of initial infection; (5) re-stimulation by local antigen presenting cells results in interferon-γ production and macrophage activation; (6) Failure to clear the organism results in granuloma formation.

Francisella Tularensis History:

• 1911- Tularemia first described by McCoy- cause of “plague like disease”.

• Initially named Bacterium tulerense after the Tulare county, California.

• 1912-1925- Edward Francis- studying “deerfly-fever” and made the connection between “plaque like disease” and “deerfly fever”.

• Studied the causative agent, modes of transmission and coined the name “tularemia”.

• Nobel Prize, 1959 and the name was changed from Bacterium tularense to Franciscella tularensis.

Francisella Tularensis General Characteristics:

• Does not grow on most routine media

• Grows best on cysteine supplemented agar

  • Blood-cysteine-glucose agar- Best growth & isolation

  • Enriched chocolate agar

  • Buffered charcoal yeast extract (same used for Legionella)

• Highly infectious (BSL-2 safety precautions)

  • Due to its highly infectious nature, physicians should notify the laboratory if Francisella is suspected.

• Clinical Laboratories (operating at Biosafety level 2) will only presumptively identify F. tularensis and will then forward to BSL-3 laboratory (such as state public health laboratories) for confirmation.

Identification of F. tularensis: (Biochemically)

• relatively inert biochemically

• • Full biochemical ID not recommended for identification due to

  its infectious nature

• Presumptive identification occurs based on growth requirements and observations, microscopic morphology and few preliminary biochemical reactions.

• Final Identification includes serology methods including immunofluorescence staining, and testing with specific anti-sera.

Francisella Tularensis microscopic morphology:

• Extremely small, intracellular gram-negative coccobacillus

• Highly pleomorphic

• Stains poorly on Gram stain

  • Typically missed in direct smears from tissue specimens

Francisella Tularensis colony morphology:

• Examine plates under laminar flow hood

• Small and transparent colonies

  • Pinpoint colonies after 24 hours

  • 1 to 2 mm after 48 hours

• Facultative anaerobe

• Require cysteine for growth

Francisella Tularensis if using biochemicals:

• Nonmotile

• Oxidase, urease, indole and ornithine negative

• Weakly positive catalase

• Beta-lactamase positive

• Direct fluorescence antibody and agglutination techniques are used for identification

  • Isolates can be tested for presence of polysaccharide antigen and at least one protein antigen

• Serum antibody testing commonly used to make diagnosis if patients history and clinical manifestations are compatible with diagnosis of tularemia.

  • Antibody titer >160 in single specimen highly suggestive of F. tularensis

  • Four-fold increase in antibody titer in paired serum samples (taken 2 weeks apart) strongly indicative of active disease

Francisella Tularensis clinical significance:

• Etiological agent of tularemia, a disease also known as glandular

  fever, tick fever, and occasionally as deer fly fever, rabbit fever (zoonoses) and market men’s fever.

• Present in a wide variety of wild animals, birds, and some fish or amphibians

• Potential Bioterror Agent (Category A infectious agent).

  • Extremely virulent, disease occurs at low infective dose ( 10 colony forming unit)

  • Nationally reportable disease- presumptive ID requires notification of local health department, state health department, as well as national.

Most common reservoir of Francisella Tularensis:

• Wild rabbits, muskrats and squirrels. Ticks & deerflies are most common arthropod vectors.

• Hunters, Veterinarians, and taxidermists are at increased risk.

• Transmission occurs through either (1) bite of an arthropod (2) direct contact with an infected animal, (3) ingestion of contaminated meat or water

Ulceroglandular:

• Associated with Tularemia

• Formation of an ulcer at the site of infection, swelling of the lymph glands, fever, chills, headaches, fatigue

Osculoglandular:

• Associated with Tularemia

• Eye pain, redness of the eyes, discharge from the eye, formation of ulcer in the inside of the eyelid

Oropharyngeal:

• Associated with Tularemia

• fever, pharyngitis, mouth ulcers, vomiting, diarrhea

Pneumonic:

• Associated with Tularemia

• cough, chest pain, shortness of breath

Typhoidal:

• Associated with Tularemia

• high fever, extreme fatigue, vomiting, diarrhea, splenomegaly, hepatomegaly, pneumonia

Franscisella Tularensis infection:

• The lymph node adjacent to the site of inoculation become enlarged and necrotic.

• Organism is the blood stream cause high fever, headaches, and become systemically ill with chills and generalized aches.

• Range from mild to self-limiting to fatal include ulceroglandualr, oculoglandualr. Oropharyngeal and pneumonic form.

• The organism is destined to be Biosafety level 3. One of the most common cause of laboratory acquired infection.

F. philomiragia:

• Rare cause of pneumonia

F. novicida:

• Rare pathogen, closely related to F. tularensis

  • Rare cause of pneumonia, muscle pain, and fever

• Ways to contract Tularemia

Summary of Francisella tularensis

Legionella Pneumophila general characteristics:

• Aerobic GNR

• Requires iron salts, cysteine, and high humidity for growth

  • BCYE agar is best

  • Buffered charcoal yeast extract

• Slow grower

  • Blood cultures may require 2 weeks for growth

• Culture and testing done in Class II biological safety cabinet

Legionellae ubiquitous environment:

• warm and moist conditions prevail

• Linked with various outbreaks with water tanks/cooling systems in hospitals and other environments

• 2015 outbreaks in NYC & Bronx NY. Organisms isolated from greater than a dozen water cooling tanks throughout city.

• Recovered from lakes, streams, mud, and soil in many parts of the world

• Nosocomial infection- nebulizer filled with tap water.

• No known animal reservoir

• More than 40 species of Legionella identified, but L.pneumophila is by far the most common cause of disease in humans

Legionella Pneumophilia typical specimens:

• Bronchial washings

• Lung biopsies

• Pleural fluid

• Blood

  • Sputum not optimal choice for culture due to abundance of

    normal upper respiratory flora

Legionella Pneumophilia microscopic morphology:

• Relatively thin rods

• Do not stain well with Gram stain

  • Basic fuchsin can be substituted for safranin; counterstain for up to 3 minutes

• Small, coccobacillary and occasional filamentous forms are seen more readily on Gimenez stain

• Direct fluorescence antibody testing can be useful, but

  has low sensitivity compared to culture on BCYE agar

• Gimenez stain of Legionella pneumonphilia

Legionella Pneumophilia: Colony morphology:

• Cultures should be handles under laminar flow hood (BSC-II)

  • Buffered charcoal-yeast extract agar (BCYE) is best for isolation

• Francisella tularensis can also grow on BCYE agar and found in same respiratory specimens as L.pneumophila which is another reason why these plates should only be examined under laminar flow hood

• Visible colonies appear after 3 to 4 days incubation

• May be round or flat with entire edges, glistening, and convex

• Ground glass speckling like a shattered windshield

• Pigment varies from colorless, grayish, pale green to iridescent pink or blue

• May also be translucent

Legionella Pneumophila: Key biochemical and other reactions:

• Catalase positive

• Gelatinase and beta-lactamase positive

• Oxidase and hippurate positive

• Growth on BCYE and pale yellow-green fluorescence under long- wave fluorescent light (Wood’s lamp)

Legionella Pneumophila immunologic methods:

• Urine antigen testing

• Serum antibodies can be tested but offer low sensitivity

• Antibodies against organism slowly rise and often do not reach peak levels until 4-8 weeks after infection

• Many patients retrospectively diagnosed by indirect fluorescent

  antibody testing

• Four-fold rise in anti-Legionella antibody to a titer of 128 or higher is considered positive

Legionella Pneumophia clinical significance:

• serogroup 1 is the major cause of Legionnaire’s disease.

• Disease most common in males over age of 50, with risk factors of chronic respiratory conditions or smoking

• Asymptomatic infection possible among all age groups

• Organism commonly acquired through inhalation of aerosols created by contaminated air conditioners, humidifiers, shower- heads, and other similar sources

• Disease ranges from mild short term febrile illness, to acute purulent pneumonia, typically affecting those older individuals or immunocompromised the worst. Fatality rate is as high a 10%

• L. pneumophila can also cause Pontiac fever, an influenza-

  like illness that initially occurred during an outbreak in Michigan.

• Nationally reportable disease

Legionella Pneumophila treatment:

• Erythromycin drug of choice (penetrates WBCs to reach intracellular orgs) or Rifampin when delayed response to treatment

Legionella Pneumophila epidemiology:

• Organism is found worldwide and spread via

• Air conditioning

• Cooling towers and waters systems ( hot tubs).

• Nosocomial setting spread through nebulizer with water

• Survive in most environment and in the presence of disinfectant such as chlorine.

• Bacteria form biofilms.

Legionnaires’ disease vs. Pontiac Fever: Legionnaires’ disease

• More severe causes morbidity and death unless

  treated properly

• Incubates for 2 to 10 days followed by symptoms of

  fever, chills, dry nonproductive cough, headache

• Pneumonia is the primary manifestation

• Often present as multi-organ disease, including the GI tract, CNS, liver, and kidneys.

• Pulmonary function deteriorates in patients with no treatment

Pontiac Fever:

• Self-limiting with symptoms of fever, chills, muscle pain, general

  body weakness and lethargy (malaise), headache, flu-like

• No evidence of pneumonia

• Symptoms develop over a 12 hour period and then stick around for 2

  to 5 days, then spontaneously disappears without antibiotic treatment.

  • There have been no deaths reported

  • Hypersensitivity reaction maybe the cause of pathology

Who should be tested for Legionnaires’ disease?:

• Hospitalized patients with enigmatic pneumonia

• Patients with enigmatic pneumonia sufficiently severe to require care in the ICU

• Compromised host with pneumonia

• Patients with pneumonia in the setting of a legionellosis outbreak

• Patients who fail to respond to treatment to a ß- lactam or cephalosporin

• Patients that have traveled away from their home within two weeks before the onset of illness

• Patients suspected of nosocomial pneumonia with unknown etiology

• Legionella on BCYE agar

• Colonies are small with a crystalline- like, ground glass appearance

• L-Cysteine, soluble ferric pyrophosphate, and alpha- ketoglutarate enhances the growth of Legionella species

• Activated charcoal removes toxic metabolic products

• Protein and other growth nutrients are supplied by yeast extract

Legionella Pneumophila lab diagnosis: Culture is the gold standard: BCYE alpha

• Nonselective

• Buffered charcoal yeast extract medium suppl. with alpha-ketoglutaric acid

• Adding specific supplements like glycine, vancomycin, polymyxin B, and cycloheximide (GVPC) makes it more selective for Legionella, but it should be used alongside non-selective BCYE because some Legionella species may be missed.

• Should be used together with a non-selective BCYE because it inhibits some Legionella sp that do not produce beta lactamase. The medium is not fully selective, as other bacteria like Pseudomonas or Bacillus might still grow.

Legionella Pneumophila lab diagnosis: Culture is the gold standard: MWY (Modifed Wadowsky-Yee medium)

• includes similar additives and is another selective option( contains glycine, vancomycin, polymyxin B, and anisomycin)

• If bacteria grow on BCYEα but not on other media (e.g., BAP or BCYEα without cysteine), they may be Legionella and should be further tested.

• Confirm suspected Legionella using Direct Fluorescent Antibody (DFA) testing or by sending samples to a reference lab.

• Biochemical test are of limited use

  • Hippurate, β-lactamase production, gelatinase, oxidase, motility, autofluorescence.

Legionella Pneumophila: Flourecence:

• Detection by DFA, direct fluorescent antibody

• specific antibodies conjugated to FITC (fluorescein

  isothiocyanate)

• Fluorescence microscope

• some false-positives

• Sensitivity is low

Urine antigen test:

• Detect soluble lipopolysaccharide antigens

• High sensitivity with serogroup 1

• lower sensitivity with other serogroups and species

• Antigens persists in the urine of treated patients

• 50% are positive after 1 month

• 25% are positive after 2 months

• Immunosuppressed patients are positive for up to a year

Serology of Legionella Pnuemophila:

• Diagnosed with IFA, indirect fluorescent assay

• test for a serologic response to infection

• A level of 1:128 or greater (4-fold) is considered diagnostic

• In the first week of disease an increase in titer can be seen in 25-40% of patients

• Because high titers can persist, one positive test cannot be used to define disease

Nucleic Acid Amplification Assays (NAA or PCR): Legionella Pnuemophila:

• Highly specific and sensitive

• Not widely available

• Anticipated to be the diagnostic method of choice in the future

• False-negatives are possible

Advantages and Disadvantages of Legionella Pnuemophila tests

• Diagnostic tests for Legionella

• Urinary antigen assay AND culture of respiratory secretions on selective media are the preferred diagnostic tests for Legionnaires’ disease

Bordetella spp.

• B. pertussis

• B. parapertussi

• B. bronchiseptica

• B. avium

Bordetella samples:

• Nasopharyngeal aspirate

• Cotton swab inhibit growth of B. pertussis

  • Transport media

Bordetella Pertussis: Pertussis or whooping cough:

• In the 20th century, was one of the most common childhood diseases and a major cause of childhood mortality in the United States.

• Remains a major health problem among children in developing countries.

• very contagious and is transmitted person to person

• According to CDC, 40 percent of babies sick with whooping cough catch it from their mothers.

Pertussis vaccination:

• reemergence of pertussis among highly vaccinated children

  seems to be due to lower protection by acellular vaccines.

• Only key to preventing the disease is vaccination

• Whooping cough vaccination is a must for all

• How to acquire Whooping cough

Pertussis toxin: mechanism of action:

• PT is secreted from the bacteria and binds to a sialoglycoprotein host cell receptor,

• PT is then endocytosed and trafficked through the Golgi apparatus to the endoplasmic reticulum (ER).

• In the ER, the B pentamer binds to ATP and dissociates from the A subunit.

• The A subunit is then transported on exosomes to the cytoplasmic membrane

• There, PT ADP-ribosylates the α- subunit of heterotrimeric G proteins.

• This modification inhibits G proteins function, including their ability to inhibit cyclic AMP (cAMP) formation.

• The result of these modifications is high cAMP levels and an overall inhibition of immune cell recruitment to the site of infection

Bordetella Pertussis: General Characteristics:

• Etiologic agent of Whooping cough

• Humans only known source of the organism

• Nonmotile, strict aerobe requiring rich medium for growth

• Best cultured on charcoal-containing medium

• Bordet-Gengou can also be used (potato-sheep blood- glycerol including penicillin)

  • Regan-Lowe used for transport

• Requires 3 to 7 days incubation in moist container

• Faintly stains with Gram stain

• Most cases occur in children under age of 5, and most deaths in infants.

• Transmission by inhalation of contamination aerosols from individuals with early stages of disease

• Communicability rates of 30-90%

• After 2 weeks- “catarrhal stage” occurs where patient only has symptoms of mild cough and sneezing, but is highly infectious with large number of organisms in the respiratory droplets

• Explosive “paroxysmal stage” follows with severe cough and “whooping” sound during inhalation

Bordetella Pertussis useful biochemical tests:

• Biochemical tests usually not performed in clinical laboratory

  • Acid, but no gas, from glucose and lactose

Bordetella Pertussis: Microscopic morphology:

• Very small, gram-negative coccobacilli

• Singles or pairs

• Faintly stains with Gram stain

  • can use basic fuchsin or safranin O as counterstain

Bordetella Pertussis colony morphology:

• Bordet-Gengou medium with 20% blood

• Diffuse zone of hemolysis after 7 days

• Smooth, mucoid, and convex

• Shiny “Mercury drops” with a pearly luster

• White colonies with a mother-of-pearl opalescence on Regan Lowe medium

Bordetella Pertussis key reactions:

• Specimens included nasal swab or nasopharyngeal swab

• Biochemical testing not routinely utilized

• Outdated practice included “Cough Plate” inoculation- where an agar plate would be inoculated by droplets expelled during paroxysmal coughing (No longer performed).

• Identify with:

• DFA and slide agglutination tests with culture

• Direct fluorescent antibody (DFA) must be performed in tandem with culture due to low sensitivity (50%)

• Serologic Tests with antibodies not very useful for rapid diagnosis because agglutination properties do not appear until third week of illness

• Molecular PCR- most sensitive method of ID

Bordetella Pertussis: Treatment:

• Erythromycin effective when administered by catarrhal stage, or as prophylaxis for unimmunized patients. Resistance has been reported

• The recommended antimicrobial agents for treatment or chemoprophylaxis of pertussis are azithromycin*, clarithromycin, and erythromycin. Clinicians can also use Trimethoprim-sulfamethoxasole. Clinicians should choose the antimicrobial after consideration of the:

  • Potential for adverse events and drug interactions

  • Tolerability

  • Ease of adherence to the regimen prescribed

  • Cost

Bordetella Pertussis: Immunization:

• Several vaccines available and are usually administered with toxoids from Corynebacterium diptheriae and Clostridium tetani, otherwise known as the DTaP vaccine

• Infants should receive three injections of vaccine during first year of life, followed by two booster injections

• Adults who do not receive booster immunization have decreased immunity and are primary reservoir for the organism.

• Nationally reportable disease

Bordetella Pertussis: Antigenic and biologically active products:

• • Pertussis toxin

  • Filamentous hemagglutinin (FHA)

  • Agglutinogens

  • Adenylate cyclase

  • Pertactin

  • Tracheal cytotoxin

• These products are responsible for the clinical features and an immune response to one or more produces immunity following infection. Some observational studies suggest that pertussis infection can provide immunity for 4 to 20 years, but that it is not life-long.

• Bordetella spp. Oxidase & Urea

Brucella: General Characteristics:

• Intracellular bacteria

  • usually found within animals, humans accidental hosts

  • DNA analysis identified only one true species, B. melitensis, with a number of biovars; however, they generally are still referred to by original “species” designation.

• Four biovars known to infect humans, with typical animal host for each are: B.melitensis (goats and sheep), B.suis (swine), B.abortus (cattle), and B.canis (dogs)

• Strict aerobe

• Complex growth requirements

  • amino acids, vitamins, salts, and glucose for culture continuum

  • For direct isolation from patients, trypticase soy agar or blood culture medium is used.

• Slow growing on BAP or Choc; incubation in presence of 5-10% CO2

  • B. abortus is only biovar with strict requirement for CO2

Brucella: Microscopic morphology:

• GNR

• Stain irregularly and pale with Gram stain

• Coccobacillary forms dominate in early cultures

• Bipolar staining sometimes observed

Brucella: Colony morphology:

• Small, smooth, convex, transparent and nonhemolytic colonies on enriched medium after 3 to 5 days of incubation

Brucella: Useful biochemical tests:

• Relatively inactive metabolically

  • Catalase positive

  • Oxidase positive

  • Urease positive

  • Many strains also H2S +

  • Reduces Nitrates to nitrites.

Brucella: Key biochemical and other reactions

• Presumptive ID with Gram stain, and positive oxidase and urease (Most clinical laboratories)

• H2S, CO2 requirement and susceptibility to dyes used to identify biovars (Reference laboratories)

• Serological testing may offer additional basis of identification

• IgG titers >80 indicates active infection

• If titers negative & strong clinical presentation of brucellosis suggested, additional tests may be performed to determine “blocking antibodies” of IgA class.

Brucella: Clinical Significance:

• Causative agent of undulant fever

• In animals, the brucellae localize in the pregnant uterus causing “contagious abortion” (erythritol).

• Highly invasive organism spreads to other animals or humans in

  contact with aborted fetus. Organism can also survive in dry soil up

  to 60 days

• Ingestion of unpasteurized milk or cheeses and other products that contain unpasteurized milk is an important route of transmission for in humans

• Brucellae localize in mammary glands in animals and can be shed in

  milk for years

• Farmers, veterinarians, and slaughterhouse workers are at greatest risk (zoonoses) but is also the most common laboratory acquired

  infection

• Potential bioterror agent

  • Nationally reportable disease

  • Appropriate authorities must be notified from local health departments, through state and national laboratories. Original specimens must be preserved for possible criminal investigation.

Brucella: Pathogenicity

• Fever, malaise, weakness and nonspecific aches pains major

  manifestations of brucellosis. Different degrees of disease severity are

  associated with different biovars.

• Incubation period ranges from 1 to 6 weeks, with slow insidious onset of disease. Bacteria disseminated via the lymphatics and bloodstream to many parts of the body.

• Granulomatous nodules form, and may progress into abscesses- Bone marrow.

• CNS symptoms, and lymph node enlargement commonly noted as well

• Undulant fever- daily periodicity with extreme sweating associated

• Generalized symptoms usually subside over period of weeks to

  months, although localized symptoms may persist longer, even for

  years.

Brucella: Treatment:

• Immunity after recovery, although reinfection is possible

  Brucellae usually susceptible to tetracycline, streptomycin, and

  ampicillin.

• Intracellular nature of organisms make them difficult to eradicate;

  extended administration of tetracycline may be required.

Brucella: Prevention:

• If brucellosis is suspected in a patient, clinicians should note “suspect or rule out brucellosis” on the laboratory submission.

• The best way to prevent brucellosis infection is to be sure you do not consume:

  • undercooked meat

  • unpasteurized dairy products, including:

  • milk

  • cheese

  • ice cream

  • Pasteurization is when raw milk is heated to a high temperature for a short period of time.

  This heating process destroys harmful bacteria that may make the milk unsafe to consume.

  • If you are not sure that the dairy product is pasteurized, do not eat it.

  • People who handle animal tissues (such as hunters and animal herdsman) should protect themselves by using:

  • rubber gloves

  • goggles

  • gowns or aprons

  • This will help ensure that bacteria from potentially infected animals do not get into eyes or inside a cut or abrasion on the skin.

Brucellosis:

• Common laboratory acquired infection

  • must work in Biosafety cabinet

  • avoid aerosol

  • never sniff the plate!

HACEK Group:

• Haemophilus parainfluenzae

• Aggregatibacter

• Cardiobacterium

• Eikenella

• Kingella

HACEK group: general characteristics:

• Group of fastidious Gram negative coccobacilli organisms

• Slow growing Gram negative Coccobacilli

  • Hold blood cultures up to 2 weeks to isolate

  • Require or stimulated by CO2

HACEK: Clinical significance:

• Associated with 3% subacute bacterial endocarditis (SBE)

  cases

• Most cases of SBE caused by viridans streptococci

• May play a role in formation of fatty plaque walls in arteries

• Occasionally implicated in infections other than SBE

• Usually part of the normal oral flora

• Infections occur due to poor oral hygiene, dental manipulation

  and damaged heart valves are predisposing factors

H. parainfluenzae:

• described previously with Haemophilus spp. Normal upper

  respiratory flora.

  • Requirement of NAD (factor V) for growth (NG on BAP)

Aggregatibacter:

• A. aphrophilus (formerly known as H. aphrophilus)

  • Coccobacillary

  • Round, convex colonies

  • Requires CO2

  • Requires heme (factor X) for initial isolation (grows on BAP)

  • Can be weakly ALA positive

  • Nonhemolytic on horse blood agar

  • Weak oxidase positive

  • Catalase, indole and urease negative

  • Glucose and lactose positive

Aggregatibacter actinomycetemcomitans: Macroscopic colony morphology:

• • Rough colonies on TSBY agar (Tryptic Soy Broth Yeast Extract agar)

  • Irregular edges, rough surface, star-shaped internal structure,

  pitting on NA (nutrient agar).

  • Smooth colonies on subculture

  • Requires CO2

A. actinomycetemcomitans: biochemicals:

• Oxidase and catalase positive

  • Indole negative

  • Glucose and maltose positive

A. actinomycetemcomitans: Clinical significance:

• Associated with periodontal disease and can cause other infections such as bacteremia and osteomyelitis.

• “Comitans” means accompanying. Aggregatibacter

• actinomycetemcomitans is frequently isolated with

• Actinomyces israelli

• actinomycetemcomitans on blood agar. Note the

  star-shaped centers of the colonies

Cardiobacterium: C. hominis:

• Facultative anaerobe

• Pleomorphic, gram variable

• Rosette or flower-like arrangement on Gram stain

• Alpha-hemolysis

Cardiobacterium: C. hominis: Biochemicals:

• Oxidase, nitrate and indole positive

  • Catalase negative

  • Glucose, maltose, sucrose positive

  • Lactose negative

  • It is often seen in patients with underlying heart valve abnormalities, such as those with prosthetic heart valves or congenital heart defects.

Eikenella: Eikeella corrodens:

• Facultative anaerobe

• Small, straight, GNR

• Small, grayish, with slight green discoloration on BAP

• Bleach-like odor

• Lemon-yellow pigment (observed on swab)

• Approximately 50% pit or corrode agar

• Most commonly associated in wounds caused by human bites or “fistfights”

• Also isolated from skin infections of drug addicts who lubricate needles with saliva before injecting drugs

Eikenella corrodens: Biochemicals:

• Oxidase and nitrate positive

• Catalase, urease, and indole negative

• Carbohydrate fermentation negative

Kingella:

• K. kingae, K. dentrificans, K. oralis (oral cavity and upper respiratory commensal)

Kingella kingae: general characteristics:

• Short, plump rods with blunt ends

• No growth on Thayer-Martin (K.denitricans will)

• Growth on Sheep blood agar requires several days in 5-10% CO2

• Beta-hemolytic on SBA

• Variable colony; Some appear as “fried-egg” with haze around center and pits agar

Kingella kingae: biochemicals:

• • Oxidase positive

  • Most are nitrate positive

  • Catalase negative

  • Glucose and maltose positive

• Selected characteristics of HACEK group

Additional Isolates:

• Capnocytophaga

  • Streptobacillus

  • Bartonella

  • Pasteurella

Capnocytophaga:

• Normal flora of humans, dogs, cats

• C. ochracea, C.sputigena, C. gingivalis, C. canimorsus

• Associated with dogs and cats bites

• Can cause fulminant sepsis with disseminated intravascular coagulation (DIC)

• Also cause of periodontitis, septicemia, endocarditis

• Isolated together with actinomycetes (like A. actinomycetemcomitants)

Capnocytophaga: General Characteristics:

• Normal flora of mouth and oral cavities

• Gram negative, fusiform rods

• Colonies exhibit a characteristic gliding motility on agar

  • Similar to that of Proteus spp. but not as extensive

• Require increased carbon dioxide for growth

• Non-hemolytic and produce a yellow-orange pigment

Capnocytophaga: biochemical reactions:

• • Negative for most biochemical reactions

  • Hydrolyze esculin and reduce nitrate

Capnocytophagoa: Seven species:

• Dog and cat

  • C. canimorsus and C. cynodegmi

    • Oxidase and catalase positive

• Human

  • C. gingivalis, C. ochracea, C. haemolytica, C. sputigena and C. granulosa

    • Oxidase and catalase negative

Capnocytophaga: Clinical Manifestations:

• Produce an immunosuppressive factor (protease)- virulence

  factor

• Most common isolate is C. ochracea

• Juvenile periodontal disease, endocarditis and oral ulcers in

  granulocytopenic individuals

Capnocytophaga: Laboratory diagnosis:

• Slow growth on sheep blood and chocolate agars with 5-10%

  carbon dioxide at 35-37°C

• No growth on MAC

• Blood cultures may take up to 7 days for positive results

• Presumptive identification

  • Yellow-pigmented, spreading colony

  • Thin, fusiform, gram negative rods

  • No growth in ambient air

Streptobacillus S. moniliformis general characteristics:

• Microaerophile

• Pleomorphic GNR forming long irregular chains

• Best growth:

• Medium supplemented with serum, protein, egg yolk or starch

• 37°C (organism fail to grow at 22)

• “L forms” are readily observed in most cultures

• Gram negative bacteria that have lost most of their cell wall, but retained outer membrane and peptidoglycan that are still able to grow and divide

L-form:

• Loss of cell wall

• Long irregular form

• “Lesion” in French- initially described as due to abnormal shape in culture.

Streptobacillus: Useful biochemical and other tests:

• Nonmotile; lacks a capsule

  • Oxidase, catalase, indole, and nitrate negative

  • Serology for serum antibodies (reference laboratories)

Streptobacillus Microscopic Morphology:

• String of beads or pearls

Streptobacillus: Colony morphology:

• • BHI agar with 20% horse serum:

• Small, colorless to grayish, smooth and glistening

  • Irregular edges possible

  • “L forms” resemble fried eggs

  • BA: Cottonlike colonies after 3 days incubation

  • Broth: “fluff balls” or “bread crumbs” at bottom of tube

Streptobacillus clinical significance:

• Human infection usually occurs following a rat bite, resulting in a

  disease known as rat-bite fever ( also caused by spirillum minus)

  • Normally found in throats of rats, mice, guinea pigs and other rodents

  • When infection occurs via ingestion of contaminated milk, the disease is known as “Haverhill fever”.

  • Fever and petechial or blotchy rash present on extremities after 2 day incubation.

  • Painful polyarthritis involving knees, ankles, elbows, and other joints in 50% of patients

• May cause false positives on VDRL screening test for syphyllis

Bartonella: General characteristics:

• Facultative, intracellular gram negative coccobacilli