Gram-Positive Bacilli: Corynebacterium

Gram-Positive Bacilli

• Corynebacterium is a non-spore-forming, Gram-positive bacillus that causes bacillary infections.
• Pathogenic organisms include bacteria, viruses, parasites, and fungi.
• Gram-positive and Gram-negative bacteria can be aerobic and anaerobic.
• Pathogenic Gram-positive bacteria are classified as either cocci or bacilli.
• Cocci include Staphylococcus, Streptococcus, and Enterococcus.
• Bacilli include Bacillus, Clostridium, Listeria, Corynebacterium, Nocardia, and Actinomyces.

Corynebacterium Species: General Characteristics

• Corynebacterium species are Gram-positive, non-spore-forming rods.
• The bacteria arrange in palisades which form "L-V" shapes or "Chinese characters".
• They are pleomorphic, with "club-ends" and have a beaded appearance due to metachromatic granules.

Other Attributes of Corynebacterium sp

• Corynebacterium is closely related to Mycobacteria and Norcardia, due to slight acid fastness and a common cell wall structure.
• Corynebacterium are facultative anaerobes but grow best in aerobic conditions.
• These require complex growth nutrients, including 8 essential amino acids.
• Selective and differential media for Corynebacterium include Loffler Medium, Telluride blood agar, and Modified Tinsdale medium.
• Corynebacterium ferments glucose and maltose, producing acid and gas.
• Corynebacterium are found as free-living saprophytes and are members of the usual flora of humans and animals.

Significant Corynebacteria Species

• Corynebacterium diphtheriae is the most significant pathogen, causing diphtheria.
• Other Corynebacterium species may cause infections in immunocompromised hosts.
• Less significant Corynebacterium species include C. xerosis, C. pseudodiphtheriticum, C. pseudotuberculosis, C. jekeium, and C. ulcerans.

C. diphtheriae: Agent of Diphtheria

• Toxigenic Corynebacterium diphtheriae has a worldwide distribution but is rare in places with vaccination programs.
• The 3 biotypes of C. diphtheriae are var mitis, var gravis, and var intermedius; there is also an additional biotype called var belfanti.
• Var gravis causes the most severe form of the disease.
• The exotoxin is also known as diphtheria toxin which is a major virulence factor.
• The toxin is produced by lysogenic strains and are lysogenized by bacteriophage with a toxin gene (tox+).
• The toxin is antigenic.

Virulence Factors

• The components that give it the capability of establishing infection are local tissue invasion and rapid growth.
• It can cause a quick toxin elaboration potential.

Bacteriophages

• A lysogenic strain is lysogenized by a bacteriophage with toxin gene (tox+).
• The lytic cycle of a bacteriophage involves attachment, entry of phage DNA with degradation of host DNA, synthesis of viral genomes and proteins, assembly, and release.
• Some phages may follow a lytic or lysogenic pathway.
• In lysogeny, phage DNA is integrated into the bacterial genome; bacteria live.
• Lysogenic bacterium is immune to further infection.

Lysogenization in C. diphtheriae

• Lysogenization involves the insertion of a dtx gene in viral DNA into Corynebacterium diphtheriae.
• Excretion of DT happens in absence of Fe.
• Expression or Repression in absence of Fe.

Toxin of Corynebacterium diphtheriae

• The toxin of Corynebacterium diphtheriae consists of two fragments: A and B.
• The A-Fragment, the active fragment inhibits protein synthesis by inactivation of EF2.
• EF2 is required for elongation of a polypeptide chain on ribosomes.
• The toxin catalyzes a reaction between ADPR and EF2 to form an ADPR-EF complex, which then inhibits protein synthesis.
• The A-Fragment leads to cell or tissue death, potentially through apoptosis.
• The exotoxin's mode of action is similar to that of Pseudomonas aeruginosa.
• The B-Fragment is the binding fragment, and binds to specific cell membrane receptors.
• The receptors are heparin-binding epidermal growth factor and are rich on cardiac and nerve cells.
• The b-fragment is what mediates the entry of fragment A into the cell.
• The mw of corynebacterium diphtheriae is 62,000 Dalton and is active at 0.1 μg/kg.
• It is secreted in inactive form and required tryptinization for conversion to toxigenically active form.
• A-B fragments are held together by disulphide bond.
• The B-fragment binds eukaryotic cell membrane and mediate entry of A-fragment into the cell

Cell Death

• Release of enzymatically active A fragment in cell surface results in protein synthesis inhibition and cell death.

In-vitro factors affecting C. diphtheriae Toxin Production

• Low iron(Fe) concentration, low osmotic pressure, amino acid types and concentration, and pH-5.1 affect toxin production.
• Suitable carbon source- glucose, citrate and succinate affect toxin production.
• Suitable nitrogen source- ammonium compounds affect toxin production.

Transmission and Risk Factors

• The reservoir is found in human carriers who are usually asymptomatic.
• The bacteria is transmitted through respiratory aerosols and skin lesions.
• The temporal pattern occurs during winter and spring
• Communicability lasts up to several weeks without antibiotics.
• Transmission occurs through respiratory secretions, spread by droplets, and through direct contact.
• Risk factors include poor nutrition, crowded or unsanitary living conditions, low vaccine coverage among infants and children, and immunity gaps in adults.

Pathogenesis & Clinical Types of diphtheria

• The incubation period for diphtheria is 2-5 days, with a range of 1-10 days, and may involve any mucous membrane.
• The types of diphtheria are classified based on the site of infection, including respiratory, ocular, cutaneous, and genital diphtheria.
• Respiratory diphtheria includes pharyngeal, tonsillar, laryngeal, and anterior nasal infections.
• Cutaneous diphtheria manifests as local disease and may include any area of the skin.
• localized pharyngeal and tonsillar diphtheria are most common and have insidious onset.
• Multiplication in epithelial cells will trigger inflammation.
• Toxin release = Necrosis + exudate formation.
• Exudate spreads within 2-3 days and may form adherent pseudomembrane.
• the pseudomembrane is fibrin, bacterium, and inflammatory cells, no lipid
• Pseudomembrane may cause respiratory obstruction.
• Fever is usually not high but patient appears toxic.
• There can be systemic Pharyngeal Diphtheria because the toxin is absorbed in the blood stream and carried systemically.
• There can be distant complications of a localized disease.
• This causes neuropathy and motor neuropathy.
• Paralysis of soft palate from 3rd week is common
• It can affect the heart, nervous system and kidneys.
• Myocarditis can then cause Cardiac arrythmias or Congestive cardiac failure and Sudden death.
• Cutaneous Diphtheria is prevalent in the tropics and these infections generally occur at the site of minor abrasions.
• It may also be super infected with Streptococcus pyogenes or Staphylococcus aureus.
• It is a localized disease similar to localized pharyngeal disease
• Systemic disease is uncommon because there will generally be low toxin absorption

Diphtheria Prevention and Treatment

• Diphtheria toxoid is formalin-inactivated.
• The schedule is three or four doses + booster annually.
• It has an Efficacy of Approximately 95% and lasts for duration Approximately 10 years.
• It should be administered with tetanus toxoid as DTaP, DT, Td, or Tdap.
• Routine DTaP Primary Vaccination Schedule are divided in doses from Primary 1-4 and Booster 1-3.
• Primary 1 happens at 2 months.
• Primary 2 happens at 4 months.
• Primary 3 happens at 6 months.
• Primary 4 happens at 15-18 months.
• Booster 1 happens at 4-6 years.
• Booster 2 happens at 11-12 years.
• Booster 3 happens Every 10 years.
• Diphtheria antitoxin is produced in horses, first used in the U.S. in 1891 and is only used for treatment of diphtheria.
• They will neutralize only unbound toxin and lasts in Ab for around 15 days – 3 weeks,
• You must Wait 3-4 weeks before giving toxoid and can Only give once.

DIAGNOSIS

• Primarily Clinical: includes sore throat, fever, dypnoea, oedema and a pseudomembranous material in the upper respiratory tract

Laboratory Diagnosis

• Includes Microscopy, Culture and Isolate characterization
• Includes Demonstration of toxin production via In-vitro Tests such as Elek test, Modified Elek Test, PCR for tox gene or ELISA for toxin , as well as In-vivo Test to identify Guinea pig inoculation and Obsolate

Diagnosis

• The diagnosis mostly consists of laboratory diagnosis because the disease is confirmed via Microscopy.
• The Microscopy consists of using Grams & Alberts (Alkaline methylene blue) Stains.
• You can also diagnose them via Culture with BA, TBA, Loeffler Slant & Modified Tisndale Medium.

Bacteriology

• Gram-positive aerobic are arranged in V or L shapes like “Chinese letters”
• metachromatic granules are seen on Albert's stain
• C. ulcerans and C. pseudotuberculosis can also be toxigenic
• Must be distinguished from “diphtheroids”

Management

• Management is considered a Medical & Public Health Emergency!
• This requires Clinical diagnosis while you Seek expert advice to treat with Antitoxin & Antibiotics followed by Supportive measures.
• Contacts or Carriers can be treated via Antibiotics & Toxoid boosters with Surveillance, screening and isolation.
• Antitoxin only if symptomatic
• Drugs of choice consist of Penicillin, erythromycin or gentamicin

Control

• In order to have better Sanitary, you need to reduce carrier rate by use of vaccine.
• Immunological involves Active immunization with toxoid in DPT, used for the uninfected
• And Passive immunization with antitoxin can be used for patients.