Cornyebacterium

Corynebacteria

Non-spore-forming,non-branching, catalase positive bacilli

Cornyeform or Diptheroid

They are slightly curved , gram-positive rods that may occur in singly or in palisades, with irregular swelling at the ends “club shaped” this termed as _______ or ______, diphtheria like bacteria.

Skin and mucous membranes

Normal Flora

Lipophilic and Nonlipophilic

2 classifications

• Corynebacterium urealyticum

• Corynebacterium jeikeium

Lipophilic Spp.

• fastidious and slow growers

• Require > 48 hours to grow

Characteristics of Lipophilic spp.

Addition of lipids :

• Tween 80

• Serum

These enhances the growth of your bacteria in the culture media

• Corynebacterium ulcerans

• Corynebacterium pseudotuberculosis

• Corynebacterium striatum

• Corynebacterium xerosis

• Corynebacterium pseudodipheriticum

Nonlipophilic spp

• often dismissed as commensals

• Can be considered opportunistic pathogens when they are isolated in various body sites

Characteristics of nonlipophilic spp.

Corynebacterium diptheriae

Most significant pathogen of the group

• “chinese letters” or “picket fence appearance”

Kleb-Loeffler’s Bacilli

Corynebacterium diptheriae is also known as ?

Chinese letters or picket fence appearance

Microscopic morphology

Bacilli, Club-shaped

Shape

Gram positive

Gram-staining reaction

Non-branching

Structure

Non-spore forming

Composition

Non-motile

Motility

Catalase positive bacilli

Catalase reaction

Aerobic

Oxygen tolerance

Pleomorphic

The ability to alter their shape in response to environmental conditions

Penicillin

Drug of choice

Erythromycin

Drug of choice for individuals resistant to penicillin

Prompt administration of Antitoxin

Treatment

Vaccination

Most patients don’t develop immunity after infection with this bacteria, _________ should be administered after a patient recovers.

• Formalin treated

• part of a trivalent DTP (Diptheria, Tetanus, Pertussis) vaccine

Effective toxoid vaccine

DTP (Diptheria, Tetanus, Pertussis)

Produces antibodies against the Diptheria toxin Only the disease not the Infection.

Diptheria Toxin

This is the Major virulence factor of the bacteria

It is an exotoxin produced by strains of the C. dipgeriae that are infected with a lysogenic beta-phage that carries the tox gene.

Lysogenic beta-phage

Virus infecting the C. diptheriae

• Oxygen

• Alkaline pH

• Iron concentration in medium

Environmental Conditions needed for this bacteria to grow outside the body (Toxin production : In vitro)

7.8 - 8.0 pH

Alkaline pH

Iron concentration in medium

The amount of iron needed for optimal toxin production is less than the amount needed for optimal growth.

Fragment A and Fragment B

Diptheria toxin is a protein made up of two polypeptide chains namely ______ and ______.

Fragment A

• Enzymatically active site

• Responsible for cytotoxicity

• This disrupts protein synthesis

Fragment B

• Receptor binding site

• Mediates (assists) the entry of fragment A into the cell cytoplasm

• Respiratory Diptheria

• Cutaneous Diptheria

Clinical Infection

Respiratory Diptheria

Is the most common clinical infection and it occurs in the non-immunized populations , carried/ infects the upper respiratory tract.

• tonsils & pharynx

Inhalation of droplets containing the bacteria or via hand-to-mouth contact

Infection of Respiratory Diptheria

2-5 days

Incubation period of Respiratory Diptheria

Low grade fever, Malaise, Mild sore throat

Symptoms of Respiratory Diptheria

The organisms infects epithelial surface —> Bacteria multiply and produce toxins —> cause cell necrosis and inflammation —> formation of greyish-white pseudomembrane —>

If membrane spreads —> suffocation

Toxins observed —→ systematic effects:

• Kidney (damage)

• Heart (death)

• Nervous system (Demyelinating peripheral nephritis )

Pathogenisis of Respiratory Diptheria

Cutaneous diptheria

Uncommon, prevalent in the tropics and may have toxigenic and nontoxigenic strains

• usually occurs in patients with poor hygiene

Wounds/skin of infected persons

Where is Cutaneous diptheria found?

They have non-healing ulcers with a grey membrane

Appearance of Cutaneous diptheria

Babes Ernst granules,

Methylene blue

In Microscopy, this Contains metchromatic granules called _________that have a rod beaded appearance especially when stained with _________.

Letter V or Letter L

Very pleomorphic so they have varying shapes that may appear in palisades or in sharp angles to one another in the ________ or _______ formation.

Babes-Ernst Granules

Represent the accumulation of polymerized polyphosphates

• Sheep’s Blood Agar

• Loeffler’s Medium

• Cystine-tellurite blood agar

• Tinsdale agar

Culture medium

37 degrees celsius,

Can also grow at 150-40 degrees celsius

In culture, it optimally grows at what temperature?

From a Dacron swab

• nose or throat

• Cutaneous diptheria on suspected lesions

Specimen used in culture

• Small grey granular colonies

• Irregular edges

• May have very small zones of beta-hemolysis

C. dipheriae in Sheep’s blood agar

Loeffler’s Medium

• Contains Serum + Egg

• Poached-egg colonies

• Small, smooth, slightly raised greyish colonies within entire margin

• No longer recommended due to the overgrowth of normal flora

Enhances the formation of Metachromatic granules

Purpose of serum and egg in Loeffler’s medium

CBTA ( Crystine-tellurite blood agar)

• Best medium for Corynebacterium

• Produces black or brown colonies only C. diptheriae

Due to the reduction of Potassium Tellurite

Why do CBTA ( Crystine-tellurite blood agar) produce black or brown colonies ?

C. ulcerans and C. Pseudotuberculosis

These corynebacteria produce brown or black colonies with a brown halo due to cystinase activity

C. diptheriae

Lacks urease production unlike C. ulceran and C. pseudotuberculosis

Tinsdale agar

Produce grey/black colonies surrounded by a dark brown halo (forming hydrogen sulfide)

urease Negative

Urease test result for C. diptheriae

• Magenta or pink

• > 8.4 pH

Positive urea test

Urease test

Tests for the presence of the urease enzyme

Phenol Red

pH indicator for urease test

Christensen’s Urea broth

Broth used for Urease Test

Catalase test

test to determine if the bacteria produces catalase enzyme by breaking down Hydrogen Peroxide into water and gas

Gas bubbles or effervescence

Positive result for Catalase test

Sugar Fermentation Test

C. diptheriae ferments glucose and maltose without gas

Remains red

Negative result for Sugar fermentation test

Change to yellow

Positive result for Sugar fermentation test

Nitrate Reduction

C. diptheriae reduces nitrate into nitrite

• test for the bacteria’s ability to use nitrate as an electron acceptor at the electron transport chain

Change from yellow to red

Positive result for Nitrate reduction test

• gravis

• Intermedius

• Mitis

Orer Corynebacterium diptheriae biotypes

Gravis

Short rods, few granules, some degree of pleomorphism

Intermedius

Long forms, poor granulation, pleomorphism

mitis

Long curved, prominent granules, pleomorphism

Guinea Pig Lethality Test

Tests for Toxigenicity, In Vivo

• ELISA

• Immunochromatographic strip assay

• PCR

• ELEK TEST

Tests for Toxigenicity, In Vitro

Guinea Pig Lethality Test

Test is (+) if the unprotected guinea pig dies within 24-96 hours and the control guinea pig lives ( a guinea pig is injected with a culture)

ELISA

Fast and convenient method to detect serum anti toxin

Immunochromatographic strip assay

Detects diptheria toxin in a matter of hours

Polymerase Chain Reaction

Detects diptheria toxin gene and can be used directly on specimen

ELEK Test

Performed for definitive ID of diptheria as a pathogen

Trypticase soy Agar with Low iron content

Culture medium used for ELEK test

Shick Test

• Test for Immunity

• A deactivated form of the antitoxin is injected intradermally

• Positive (+) lack of antibodies = redness or swelling

• No swelling = patient is still immune

• C. amycolatum

• C. urealyticum

• C. minutissimum

• C. pseudotuberculosis

• C. ulcerans

• C. jeikeium

• C. Pseudodiptheriticum

• C. striatum

• C. xerosis

Other Corynebacterium species

C. amycolatum

• One of the most frequently recovered from human specimens

• Often misidentified as C. striatum, C. xerosis and C. minutissimum

• Part of normal skin microbiota

Prosthetic joint infections, blood stream infections, endocarditis

C. amycolatum associated infections

Flat, dry, waxy , Nonlipophilic

C. amycolatum colony morphology

Resistant to B-lactams, fluoroquinolones, macrolides, clindamycin, Aminoglycosides

C. amycolatum Antibiotic resistance

Immunocompromised patients

C. amycolatum is common in?

Corynebacterium urealyticum

One of the most frequent isolated clinically significant corynebacteria

• primarily described as a urinary pathogen

• Causes UTI, bacteremia, endocarditis and wound infections

• Lipophilic and is a strict aerobe

C. urealyticum

Nitrate negative , catalase positive, rapidly urease positive within minutes following inoculation to Christensen urea broth/slant

Vancomycin

Drug of choice for C. urealyticum

C. minutissimum

Etiologic agent of Erythrasma

• superficial infection of the axillary and pubic skin

• Patches of erythrasma are initially pink but quickly depress to be brown and scaly as skin starts to shed

erythromycin or azithromycin

C. minutissimum Drug of choice

C. pseudotuberculosis

• rarely causes disease in humans

• A veterinary pathogen

• Causes Granulomatous lymphadenitis

Brown halo

C. Pseudotuberculosis in CBTA

small, yellowish-white colonies

C. Pseudotuberculosis In SBA

C. pseudotuberculosis

Urease positive , negative gekatin hydrolysis test

Penicillin, and erythromycin

Drug of choice for C. pseudotuberculosis

C. ulcerans

Infects cattle and other animals; causes mastitis in cattle and other domestic and wild animals

• produce less amount of diptheria toxin

Ingestion of unpasteurized dairy products and contact with infected animals

How do humans acquire C. ulcerans?