Corynebacterium and Bacillus Notes

Corynebacterium

• Corynebacterium are club-shaped gram-positive, non capsulated, non-sporing, non-motile rods.
• Corynebacterium diphtheriae (or Klebs-Loeffler bacillus) shows two additional features:
• Chinese letter or cuneiform arrangement in smear (V or L shaped)- due to bacterial cells
• divide and daughter cells tend to lie at acute angles to each other. This type of cell division is called snapping type of division.

Read And Learn More: Micro Biology And Immunology Notes

Metachromatic granules present at ends or poles of bacilli (also called polar bodies or Babes Ernst bodies or volutin granules):

• They are storage granules composed of polymetaphosphates.
• They are better stained with special stains, such as Albert’s, Neisser’s and Ponder’s stain.
• Granules are well developed on enriched media, such as blood agar or Loeffler’s serum slope.

Virulence Factor (Diphtheria Toxin)

Mechanism of Action

• DT is the primary virulence factor responsible for the diphtheria:
• It has two fragments: Fragment A (active unit) and B (binding unit)
• Fragment B binds to the host cell receptors (such as epidermal growth factor) and helps in entry of fragment A.
• Fragment A is internalized into the host cells and then causes→ ADP ribosylation of elongation factor 2(EF2)→ inhibition of EF2→ irreversible inhibition of translation step of protein synthesis→ cell death.
• Mechanism of DT is similar to exotoxin A of Pseudomonas.
• Toxin Production is Dependent on
• Phage coded: DT is coded by β corynephage carrying tox gene.
• Iron concentration: Toxin production depends on optimum iron concentration (0.1 mg per liter). Higher levels of iron inhibit toxin synthesis by up regulating DT repressor gene in the bacterial chromosome.
• DT repressor gene (DtxR) is an iron dependent negative regulator of DT production and iron uptake in C. diphtheriae.
• Biotypes: Among the three biotypes of C. diphtheriae, all strains of gravis, 95–99% strains of intermedius and 80–85% of mitis strains are toxigenic.
  However, toxins produced by different biotypes are antigenically similar.
• Other species: DT is also produced by C. ulcerans andC. pseudotuberculosis.

Toxoid is used for Vaccination

• Diphtheria toxin is antigenic and antitoxins are protective in nature. However, as it is virulent, cannot be given directly for vaccination.
• Toxin can be converted to toxoid which is used for vaccination. Toxoid is a form of toxin, where the virulence is lost, retaining its antigenicity.
• Toxoid formation is promoted by formalin, acidic pH and prolonged storage.
• Park William 8 strain of C. diphtheriae is used as a source of toxin for preparation of vaccine.
• Lf unit: DT is expressed as Loeffler’s flocculating unit. 1 Lf unit is the amount of toxin which flocculates most rapidly with one unit of antitoxin.

Pathogenicity and Clinical Manifestations

Diphtheria is toxemia but never a bacteremia:

• Bacilli are noninvasive, present only at local site (pharynx), secrete the toxins which spread by bloodstream to various organs.
• It is the toxin which is responsible for all types of manifestations including local (respiratory) and systemic complications (except the skin lesions which may be caused due to the organism).

Respiratory Diphtheria

This the most common form of diphtheria. Tonsil and pharynx (faucial diphtheria) are the most common sites followed by nose and larynx and rarely non-respiratory mucosa, such as conjunctiva or vagina. Incubation period is about 3–4 days:

• Faucial diphtheria: Toxin elicits an inflammatory response that leads to necrosis of the epithelium and exudate formation leading to formation of pseudomembrane (tough leathery greyish-white coat composed of an inner band of fibrin surrounded by neutrophils, RBCs and bacteria).
• Pseudomembrane is so named, as it is adherent to the mucosal base and bleeds on removal in contrast to the true membrane which can be easily separated (Example. as in Candida).
• Extension of pseudomembrane: In severe cases, it may extend into larynx and medium-sized bronchial airways which may result in fatal airway obstruction leading to asphyxia, which mandates immediate tracheostomy.
• Bull-neck appearance: Characterized by massive tonsillar swelling and neck edema.

Cutaneous Diphtheria

• Characterized by punched-out ulcerative lesions with necrosis, or rarely pseudomembrane.
• Cutaneous diphtheria is due to the organism itself and is not toxin mediated.
• Skin lesions can also be caused by nontoxigenic strains.
• There is increasing trend of cutaneous diphtheria nowadays, especially in vaccinated children, because antitoxins present in vaccinated people cannot prevent the disease.

Systemic Complications

Neurologic manifestations (cranial nerve involvement, Peripheral neuropathy, ciliary paralysis) and myocarditis are late toxic manifestations, occurring after weeks of infection.

Laboratory Diagnosis

The diagnosis of diphtheria is based on clinical signs and symptoms plus laboratory confirmation.

• Because of risk of respiratory obstruction, specific treatment should be instituted immediately on clinical suspicion of diphtheria without waiting for laboratory reports.
• Laboratory diagnosis consists of isolation of the bacilli and toxin demonstration.

Isolation of the Diphtheria Bacillus

Specimen: Throat swab (one or two) containing fibrinous exudates and a portion of membrane
Direct smear microscopy:

• Gram stain: C. diphtheriae appears as irregularly stained club-shaped gram-positive bacilli arranged in Chinese letter or cuneiform arrangement (V or L shaped).
• It is difficult to differentiate them from other commensal coryneforms found in the respiratory tract.
• Albert’s stain is more specific for C. diphtheriae, where they appear as green bacilli with bluish-black metachromatic granules.

Culture media:

• Enriched medium: such as Loeffler’s serum slope:
• Advantages: (1) detects growth early (6–8 hrs), (2) best medium for metachromatic granules production.
• Disadvantage: As it is an enriched medium, if incubated beyond 6–8 hrs, it supports growth of other throat commensals also.
• Selective medium: such as Potassium tellurite agar (PTA) and Tinsdale medium:
• Advantage: Throat commensals are inhibited, hence they are best media for isolation of C. diphtheriae from cases as well as carriers; as the normal flora will be inhibited.
• Disadvantage: The black colonies appear only after 48 hours of incubation.

Biochemical identification:

• Hiss’s serum sugar media: C. diphtheriae ferments glucose and maltose (by all biotypes) and starch (by only gravis)
• Pyrazinamidase test: Negative for C. diphtheriae, C. ulcerans and C. pseudotuberculosis
• Urease test: Negative for C. diphtheriae; but C. ulcerans and C. pseudotuberculosis are urease positive.
• Corynebacterium is catalase positive but oxidase negative and nonmotile.

 

Fig. 3.4.5: Elek’s gel precipitation test

Toxin Demonstration

As the pathogenesis is due to diphtheria toxin, mere isolation of bacilli does not complete the diagnosis.

Toxin demonstration should be done following isolation, which can be of two types; in vivo and in vitro:

Typing of C.diphtheriae

• Biotyping (McLeod’s classification): C. diphtheriae can be typed into four biotypes, such as gravis, intermedius, mitis and belfanti based on various properties.
• Biotype belfanti is a nitrate-negative variant of mitis biotype.
• Other methods: Serotyping, Bacteriophage typing, Bacteriocin typing and Molecular typing methods, such as PFGE.

Epidemiology

Diphtheria is an endemic disease known from ancient time. However, there is declining trend of diphtheria cases in most developed as well as a few developing countries including India due to widespread vaccination coverage.

• Source of infection: Carriers (95%) are more common source of infection than cases (5%).
• Carriers may be temporary (persists for a month) or chronic (persists for a year). Nasal carriers are more dangerous due to frequent shedding than throat carriers. Incidence of carrier rate varies from 0.1 to 5%.
• Transmission is via the aerosol route, or rarely by contact with infected skin lesions.
• Reservoir: Humans are the only reservoir Vaccine or ADS Antibiotic Carrier Not effective Effective Clinical Diphtheria ADS is treatment of choice Vaccine is given for prevention Not effective (Except early stage) Cutaneous Diphtheria Not effective Effective
• Age: Diphtheria is common in children aged 1–5 years. With wide spread immunization, a shift in age has been observed from preschool to school age. Newborns are usually protected due to maternal antibodies.

Treatment

Treatment should be started immediately on clinical suspicion of diphtheria:

• Antidiphtheritic horse serum or ADS (antitoxin): It is the treatment of choice as it neutralizes the toxin.
• Antibiotics: Penicillin or erythromycin is the drug of choice. Antibiotic plays a minor role as
• it is of no use once the toxin is secreted. However, antibiotics are useful:
• If given early (< 6 hrs of infection), before the toxin release
• Prevent further release of toxin by killing the bacilli
• Treatment for cutaneous diphtheria.
• Treatment of carriers: Drug of choice is erythromycin

Prophylaxis (Vaccination)

• Active immunization is done by diphtheria toxoid that induces antitoxin production in the body.
• Protective titer of antitoxin is > 0.01 Unit/mL.
• Herd immunity of > 70% is required to prevent epidemic spread of diphtheria.
• However, vaccine is not effective for:
• Prevention of cutaneous diphtheria
• Elimination of carrier stage
• Types of Vaccine
• Single vaccine: Diphtheria toxoid (alum or formal precipitated)
• Combined vaccine: Various vaccines available are:
• DPT: Contains DT (diphtheria toxoid), pertussis (whole cell) and TT (tetanus toxoid)
• DTaP vaccine (diphtheria, tetanus, and acellular pertussis). This can be given safely to
  older children, as this form of pertussis vaccine is devoid of neurological complication
• DT vaccine protects young children from diphtheria and tetanus
• Tdap vaccine (combined tetanus, diphtheria and acellular pertussis), it has a reduced dose of the diphtheria and pertussis vaccines and is approved for adolescents and adults.
• Td vaccine (adult tetanus and diphtheria), which is given to adolescents and adults. It contains TT and adult dose (2 Lf ) of diphtheria toxoid.
• It is recommended after 7 years.
• For adults >18 years who have completed their primary vaccination schedule, a booster dose of Td vaccine is indicated once in every 10 years till the age of 65
• For adults >18 years who have not completed their primary vaccination schedule: 3 doses of Td given at 0, 1 month, and 1 year.
• Pentavalent vaccine: DPT can also be given along with hepatitis B and Haemophilus influenzae type b.

DPT Vaccine

• DPT is the preparation of choice for vaccinating infants, because:
• Infants can be immunized simultaneously against three important childhood diseases: diphtheria, tetanus and pertussis by single injection.
• Pertussis component acts as adjuvant and increases immunogenicity of DT and TT.
  There are two types of DPT:
• Plain formol toxoid (or fluid toxoid): Toxoid is prepared by incubating toxin with formalin
• Adsorbed (alum adsorbed): Alum acts as adjuvant and increases the immunogenicity of toxoid.
• Administration of DPT:
• Schedule: Under national immunisation schedule of India, total five doses are given; three doses at 6, 10 and 14 weeks of birth followed by two booster doses at 16–24 months and 5 years.
• Site: DPT is given deep intramuscularly (IM) at anterolateral aspect of thigh, (gluteal region is not preferred as fat may inhibit DPT absorption)
• Thiomersal (0.01%) is used as preservative
• Storage: DPT should be kept at 2–8°C, if accidentally frozen then it has to be discarded.
• Dose: 1 dose (0.5 mL) contains:
• Glaxo: 25 Lf (DT), 5 Lf (TT), 20,000 million (Pertussis killed bacilli)
• Kasauli: 30 Lf (DT), 10 Lf (TT), 32,000 million (pertussis killed bacilli)
• Protective titer: Following vaccination, an antitoxin titer of ≥ 0.01 unit/mL is said to be protective
• Reactions following DPT administration:
• Mild: Fever and local reaction (swelling and indurations) are observed commonly.
• Severe: Whole cell killed Bordetella pertussis is encephalitogenic. It is associated with neurological complications.
• Hence, DPT is not recommended after 6 yrs of age.
• Absolute contraindication to DPT:
• Hypersensitivity to previous dose
• Progressive neurological disorder

Schick Test

• It is a toxin-antitoxin neutralization test, obsolete nowadays:
• It was in use long back when the vaccine was introduced initially.
• The test was being done on the people before starting immunization to identify susceptible individuals.

Nondiphtheria Corynebacterium

• Clinical diphtheriae: C. ulcerans and C. pseudotuberculosis can also produce DT and produce
• clinical diphtheriae. However, they are urease positive and usually affect animals.
• C. ulcerans: Mimics respiratory diphtheria, transmitted by cow milk
• C. pseudotuberculosis (Preisz Nocard bacilli): Affect sheep and horse
• C. minutissimum: Causes cutaneous lesion called erythrasma, which gives coral red color
  under wood lamp.
• C. jeikeium: Multidrug-resistant species, causes opportunistic infection in human.
• C. parvum, e.g. of immunomodulator.

Bacillus

• Spore forming bacilli belong to two genera:
• Bacillus: They are obligate aerobes; having non bulging spores
• Clostridium: They are obligate anaerobes with bulging spores.

Bacillus Anthracis

• B. anthracis is the causative agent of an important zoonotic disease called anthrax.
• It also gained importance recently because of its ability to be used as biological weapon.
• Virulence Factors and Pathogenesis
  Pathogenesis of anthrax is due to two important virulence factors; anthrax toxin and capsule.
  

Anthrax toxin

• It is a tripartite toxin, consisting of three fragments:
• Edema factor: It is the active fragment; acts as adenylcyclase → increases host cell cyclic AMP.
• Protective factor: It is the binding fragment that binds to the host cell receptor.
• Lethal factor: It causes cell death, inhibits mitogen-activated protein kinase pathway.
• These fragments are not toxic individually, but in combination, they produce local edema and generalized shock.
• Toxin synthesis is controlled by a plasmid (pX01). Loss of plasmid makes the strain avirulent.
• This was probably the basis of original anthrax vaccine prepared by Pasteur.
• Live attenuated spore vaccine (Sterne, Mazucchi): prepared by deleting the capsule genes.

Anthrax capsule

• B. anthracis has a polypeptide capsule made-up of polyglutamate (in contrast to the polysaccharide capsule present in other capsulated bacteria).
• Capsule is plasmid (pX02) coded.
• It inhibits complement-mediated phagocytosis.
• Pathogenesis and Clinical Manifestations Anthrax is primarily a zoonosis.
• Herbivorous animals such as cattle, sheep and less often horses and pigs are affected more commonly than the carnivorous animals.
• Human Transmission: Human beings acquire infection by:
• Cutaneous mode: By spores entering through the abraded skin; seen in people with occupational exposure to animals (most common mode)
• By inhalation of spores
• Ingestion of carcasses of animals dying of anthrax-containing spores (manifested as bloody diarrhea)
  Types of human anthrax: Mainly three types:
• Cutaneous,
• Pulmonary and
• Intestinal anthrax

 

B. anthracis as an Agent of Bioterrorism

• B. anthracis has been a major agent of bioterrorism and biologic warfare such as outbreaks in Sverdlovsk in 1979 and in United States in 2001
• Pulmonary anthrax is the most common form seen in bioterrorism outbreaks.
• It occurs via inhalation of anthrax spores from contaminated animal products.

Laboratory Diagnosis (Anthrax vs Anthracoid Bacilli)

• B. anthracis can be differentiated from other Bacillus species (Anthracoid Bacilli) by the presence of following properties:
• B. anthracis is Nonmotile and Capsulated
• MC Fadyean reaction: Polypeptide capsule is seen as amorphous purple material surrounding blue bacilli when stained with polychrome methylene blue.
• Gram staining: chain of bacilli arranged in bamboo stick appearance.
• On Agar plate: Medusa head appearance colony (under low power microscope)
• Gelatin stab: Appear as inverted fir tree appearance
• Solid media with penicillin: String of pearl appearance in culture smear
• Blood agar: nonhemolytic colonies
• Selective medium: PLET media
• DFA (Direct fluorescent antibody test): Detects capsular antigen. It is used for confirmation of
  diagnosis during bioterrorism outbreaks.
• Ascoli’s thermo precipitin test: It is a ring precipitation test, detects anthrax antigens.
• Spores can be demonstrated by phase contrast microscope or use of special stains such as hot malachite green (Ashby’s method) or 0.25% sulfuric acid (spores are acid fast).
• Lipid granules can be demonstrated by Sudan black B (Burdon’s method).

Guidelines for Diagnosis of Anthrax during Bioterrorism Attacks (CDC, 2001)

Following 2001 USA attack, CDC has prepared guidelines for identification of B. anthracis during bioterrorism.

• For presumptive identification of anthrax: Any large gram-positive bacillus with morphology and cultural properties similar to anthrax bacillus
• For initial confirmation, the tests done are Lysis by gamma phage and DFA
• Further confirmation is done by PCR.

Treatment

• Anthrax can be successfully treated if the disease is promptly recognized and appropriate therapy is initiated early.
• Antibiotics for treatment: Consists of ciprofloxacin or doxycycline, plus clindamycin, and/or rifampin, for 60 days.
• Antibiotics for post-exposure prophylaxis:
  • Ciprofloxacin for 60 days and
  • Doxycycline, for 60 days or Amoxicillin, for 60 days
• Raxibacumab: It is a monoclonal antibody that neutralizes anthrax toxin (protective antigen).
• It is intended for the prophylaxis and treatment of inhalational anthrax.

Prevention

• Live attenuated, non capsulated spore vaccine (Stern vaccine): It is extensively used in animals, but not for human use.
• Alum precipitated toxoid vaccine: It is prepared from the protective antigen. It is safe and effective for human use.

Other Bacillus Species

Bacillus Cereus
It is a normal habitant of soil, also widely isolated from food items, such as vegetables, milk, cereals, spices, meat and poultry.

Manifestations:

• Food poisoning: It produces two types of toxins; diarrheal toxin and emetic toxin
• Ocular disease: Causes severe keratitis and panophthalmitis following trauma to the eye.
• Other conditions: It rarely causes systemic infections, including endocarditis, meningitis, osteomyelitis, and pneumonia; the presence of a medical device or intravenous drug use.
• Laboratory diagnosis: B. cereus is motile, noncapsulated and not susceptible to gamma phage.
• It can be isolated from feces by using selective media such as;
• MYPA (Mannitol, egg yolk, phenol red, polymyxin and agar)
• PEMBA (polymyxin B, egg yolk, mannitol, bromothymol blue and agar)
• Treatment: B. cereus is susceptible to clindamycin, erythromycin, vancomycin. It is resistant to penicillin (by producing β-lactamase) and trimethoprim.

Bacillus Thuringiensis

• It may occasionally produce food poisoning. It is also used as larvicidal agent for mosquito control.

Bacillus used as Sterilization Control

• Geobacillus stearothermophilus and subtilis: Both are used as biological controls for autoclave and plasma sterilization.
• pumilus is used as biological control for ionizing radiation.
• Bacillus atrophaeus is used for ethylene oxide sterilizer and dry heat sterlizer.